copious urinary excretion of a male syrian hamster (mesocricetus auratus) salivary gland protein...

General and Comparative Endocrinology 186 (2013) 25–32

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General and Com parative Endoc rinology

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Copious urinary excretion of a male Syrian hamster (Mesocricetus auratus )salivary gland protein after its endocrine-like release upon b-adrenergic stimulation

Ved Prakash Dubey, Subramanya Srikantan 1, Mahabub Pasha Mohammad, Wenson David Rajan,Prabir Kumar De ⇑Centre for Cellular and Molecular Biology, Council of Scientific and Industrial Research (CSIR), Uppal Road, Hyderabad 500 007, India

a r t i c l e i n f o a b s t r a c t

Article history:Received 29 September 2012 Revised 2 February 2013 Accepted 9 February 2013 Available online 27 February 2013

Keywords:LipocalinOdorant-bindingAdrenergicIsoproterenolCholinergicKidneyLCN2Allergen

0016-6480/$ - see front matter � 2013 Elsevier Inc. Ahttp://dx.doi.org/10.1016/j.ygcen.2013.02.016

⇑ Corresponding author. Fax: +91 40 27160311.E-mail address: [email protected] (P.K. De).

1 Present address: Cancer Therapy and Research CenUTHSC San Antonio, Texas, USA.

Salivary glands, although widely considered as typically exocrine, may also release specific proteins in anendocrine manner. However, endocrine release of salivary gland proteins is not generally acknowledged since the evidences are not easily demonstrable. Sub mandibular salivary glands (SMG) of male Syrian hamsters express male-specific secretory proteins (MSP; which are lipocalins) visible in SDS–PAGE ofSMG extracts, as major bands and also detectable in immunoblots of whole-saliva and urine as low MSP crossreactions. We report here that MSP is localized in acinar cells of SMG and acute treatment with isoproterenol (IPR; non-specific b1/b2-adrenergic agonist) results in consider able release of MSP in SMG- saliva. Moreov er, acute IPR treatment markedly depletes SMG-MS P in a dose- and time-dependent manner. However, MSP depleted from SMG, far exceeds that recovered in SMG-saliva. Blood, submandibular lymph nodes and kidney of IPR-treated males showed MSP crossreactions and SDS–PAGE of their urine revealed profuse MSP excretion; this was undetectable in IPR-treated-SMG-ablated males, confirmingthat a substantial amount of MSP dep leted from SMG after IPR treatment enters circulation and isexcreted in urine. Treat ments with specific b1- or b2-adrenergic agonists also reduced SMG-MSP levels and resulted in copious urinary excretion of MSP. Co-treatments with specific b1/b2-blockers indicated that above effects of IPR, b1- and even b2-agonists are very likely mediated by b1-adrenoceptors. MSP’s detection by SDS–PAGE in urine after b-agonist treatment is a compelling and easily demonstrable evidence of release into circulation of a salivary gland protein. The possibl e means (endocrine-like or otherwise) of MSP’s release into circulation and significance of its presence in saliva, blood and urine of male hamsters are discussed.

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1. Introduction

Salivary glands are typically exocrine, secreting saliva through ducts into oral cavity. They have two major cell types, acinar and ductal, that secrete salivary proteins across their apical membran eupon sympathetic (adrenergic) or parasym pathetic (cholinergic)nerve stimulation (Bedi, 1993; Gorr et al., 2005; Nater and Rohle- der, 2009; Proctor and Carpenter, 2007 ). It is debated whether salivary gland secretory proteins can also be endocrin e secreted into circulation (Grau et al., 1994; Isenman et al., 1999; Morris et al.,2009; Murphy et al., 1977, 1980; Nexø et al., 1981; Pedersen and Poulsen, 1982; Proctor et al., 1989 ). Studies, investigatin g this measured blood levels of salivary gland secretory proteins mainly in rat and mouse after gland ablation, adrenerg ic or cholinergic stimulation or aggression (Grau et al., 1994; Isenman et al., 1999; Morris et al., 2009; Murphy et al., 1977, 1980; Nexø et al., 1981; Pedersen

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ter , Department of Medicine,

and Poulsen, 1982; Proctor et al., 1989; Venkatesh et al., 2007 ).Moreove r, blood levels of transgeni c secretory proteins expresse din salivary glands after in vivo gene-trans fer were also investigated (Gorr et al., 2005; Isenman et al., 1999; Samuni and Baum, 2011;Samuni et al., 2008 ). While some studies found evidence of release into blood (Grau et al., 1994; Nexø et al., 1981; Proctor et al., 1989;Rougeot et al., 2000; Samuni et al., 2008; Venkatesh et al., 2007 ),others did not (Morris et al., 2009; Murphy et al., 1977, 1980;Pedersen and Poulsen, 1982 ). Detection of salivary gland proteins in blood requires intricate assays (Grau et al., 1994; Morris et al.,2009; Murphy et al., 1977, 1980; Nexø et al., 1981; Pedersen and Poulsen, 1982; Proctor et al., 1989; Rougeot et al., 2000; Samuni et al., 2008; Venkatesh et al., 2007 ) and similar proteins sourced into blood from non-salivary tissues can complicate measurements (Bing and Poulsen, 1979a,b; Morris et al., 2009; Proctor et al., 1991;Tuomela, 1990 ). Thus, endocrine release of salivary gland proteins is not widely acknowledged mainly due to lack of an easily demonstrable model.

In hamsters, endocrine release of salivary gland proteins isuninvesti gated and only two studies have investigated exocrine

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26 V.P. Dubey et al. / General and Comparative Endocrinology 186 (2013) 25–32

secretion from salivary glands (from male submand ibular glands;SMG) of Syrian hamsters after stimulation by adrenerg ic and cholinergic agents (Abe and Dawes, 1982; Iwabuchi and Masuhara,1992). Among these agents, IPR (a b1/b2-adrenergic agonist, which depletes secretory protein granules selectively from salivary gland acinar cells (Iwabuchi et al., 1988; Peter et al., 1999 )) released SMG-saliva of highest protein concentratio n (Abe and Dawes,1982).

We had reported that male Syrian hamsters, express abundant 20.5 (nonglycosylated) and 24 kDa (N-glycosylated) male-spe cificproteins (MSP; products of a single gene) only in SMG, which com- prise �40% of soluble SMG proteins (De, 1996; Thavathi ru et al.,1999). MSP is a lipocalin having maximum sequence identity with odorant/pher omone-binding lipocalins (Srikantan et al., 2005;Thavathiru et al., 1999 ). MSP’s sequence has single N-glycosylati onsite and mature MSP is derived from a precursor after signal-peptide cleavage , typical of a secretory protein (Srikantan et al., 2005;Thavathiru et al., 1999 ). Although, crossreactions of both forms ofMSP are detectable in whole-saliva (Thavathiru et al., 1999 ),intriguingly , low MSP crossreactions are also detectable in urine (Thavathiru et al., 1999 ) suggesting a possibility of release ofMSP into circulation. Other than a possible role in hamster chemical communicati on, MSP might be an allergen responsible for allergy to hamsters since several mammalian lipocalin s detected insalivary gland/saliva and also urine, including a putative odorant- binding lipocalin recently identified in Siberian hamster, were found to be allergens (Hilger et al., 2012; Niitsuma et al., 2004; Tor- res et al., 2012 ).

Salivary glands are now being investigated as potential target tissue for in vivo gene transfer with a goal to release therapeuti- cally important transgeni c secretory proteins into saliva as well as circulation, for both local and systemic effects (Samuni and Baum, 2011; Samuni et al., 2008 ). Investigations on release ofMSP from hamster SMG into circulation might help to understand the secretory pathways in salivary glands, which operate for systemic delivery of salivary gland proteins.

We investiga ted here the cellular localization of MSP in SMG and the effect of b-adrenergi c agonist treatments on SMG-MSP.We show here that acute b-adrenergi c agonist treatment markedly depletes SMG-MSP releasing it abundantly into circulation and saliva and this is likely to be mediated by b1-adrenoceptor s. SMG- MSP released into circulation is copiously excreted in urine wherein it is easily detectable as major protein bands in Coomassie- stained SDS–PAGE. MSP in male hamster SMG might be a useful model to study mechanis m of stimulated- release of a salivary gland acinar cell protein into circulation and saliva.

2. Materials and methods

2.1. Chemicals used for treatments

The b-agonists, (±)-isoproterenol hydrochloride (IPR), (�)-iso-proterenol (+)-bitartrate (-IPR), salbutamol sulphate , metaproterenol sulphate, dobutamine hydrochlor ide and b-blockers , DL- propranolol hydrochlor ide, metopro lol succinate and butoxamine hydrochlori de, were all from Sigma (USA).

2.2. Treatment of hamsters and collection of tissues and urine

Our Institutional Animal Experimentation Ethics Committ ee ap- proved all animal experiments. Experiments were performed ongroups containing minimum 3 Syrian hamsters (Mesocricetu s auratus; 140–160 g; �3 months old). b-agonists or b-blockers were injected ip in saline (3 mg/dose ). Males were isolated in bedding-free cages (water ad libitum ) and 30 min after a male urinated, a dose of

b-agonist was injected. Alternatively, 15 min after urination, a b-blocker was injected and 15 min later, same blocker was re-injected along with a b-agonist. Unless otherwise stated, hamsters were sacrificed 3 h after b-agonist treatment and bladder-urine was collected using syringe and SMG (pair) were excised out. Sub- mandibu lar lymph nodes were removed 45 min and 3 h after treatment. Other tissues were collected following saline perfusion 3 hafter IPR treatment of males. Tissues were weighed and frozen.When required, hamsters were maintain ed in metabolic cages topool or collect their consecutive urinations.

2.3. Collection and processing of blood from IPR-treated and untreated hamsters

To detect MSP in blood, kidneys of males were bilaterall y-ligated under anesthesia and then males were injected IPR (± isoproterenol)or left untreated. After 3 h, blood was harvested and blood-plasma separated. Blood-pla sma-pools (15 ml) from IPR-treated and untreated males were separately fractionated step-wis e at 40%, 60%,75% and 100% (NH4)2SO4 saturatio ns. Precipitated proteins of each step were dissolved in 3 ml of 20 mM Tris–HCl (pH 7.4).

2.4. Collection of SMG-saliv a

For SMG-saliva collection (Abe and Dawes, 1982; Samuni and Baum, 2011 ), anesthetized hamsters, with both SMG ducts intrao- rally cannulat ed were injected IPR (or left untreated) and saliva was collected in microfuge tubes for 3 h. In IPR-treated hamsters,all SMG-saliva (�100–150 ll) was obtained within the 1st hour and in subsequent 2 h, no increase was observed. In untreated- con- trols, no saliva was obtained in collection tubes. Trace saliva remaining within cannulation tubings (of IPR-treated or untreated)was washed out by saline and added to collection tube and total volume in it was diluted to 1.2 ml to obtain the SMG-saliva pool.

2.5. Preparatio n of tissue extracts, SDS–PAGE and Western blots

Tissues were homogenized (2.5% w/v in 20 mM Tris–HCl, pH7.4) and centrifuged to obtain tissue-extra cts (De, 1996 ). Samples were resolved in 11% SDS–PAGE and gels were either stained (with0.1% Coomassie blue R-250) or blotted and then probed using MSP antisera (De, 1996; Thavathiru et al., 1999 ). Crossreactions were detected using I-125-labeled protein-A and autoradiograph y (De,1996).

2.6. Estimation of total MSP content in SMG extract, SMG-saliv a pools and 24 h urine pools of IPR-treat ed males

MSP contents were estimated as percentage of MSP content inSMG of normal (untreated) male hamster. Equal aliquots of SMG extracts of normal males when run in the same SDS–PAGE gel show very similar MSP band intensity after Coomassie staining (not shown). For the purpose of analysis, the MSP content of all such extracts was taken as 100%. SMG extracts of IPR-treated males and dilutions of SMG-saliva pools and 24 h urine pools of IPR-treated males were run in SDS–PAGE alongside dilutions of SMG extracts of normal males (all 60 ll). Coomassie-stai ned gels were scanned and intensities of MSP bands were measured (usingImageQuan t software). MSP band intensities in lanes containing SMG extract of normal males were compared with lanes containing samples from IPR-treated males and taking into considerati on, the dilution and total volumes, the total MSP content in each sample (as percent of normal SMG extract) was estimate d. Protein estimation of SMG-saliva pools and bladder urine of males was performed using the Bradford protein assay kit (BioRad) and bovine serum albumin as standard. All values are given as means ± SD.

V.P. Dubey et al. / General and Comparative Endocrinology 186 (2013) 25–32 27

2.7. Immunohist ochemistry

For immunohistoc hemistry, 5 lm sections of formalin-fixedSMG were processed (Paliwal et al., 2006 ) and probed with MSP antisera (1:10,000). Detection was done using peroxidase-con ju- gated anti-rabbit IgG and diaminoben zidine/H 2O2. Sections were counterstained with hematoxylin.

3. Results and discussion

3.1. Male-specific expression and cellular localization of MSP inhamster SMG

Representat ive SDS–PAGE profiles depicted in Fig. 1A show that SMG extract of male hamsters (lane 2) contain major 24 and 20.5 kDa MSP bands, which are undetect able in females (lane 4).

Fig. 1. Male-specific expression, cellular localization and IPR-induced release of SMG-MSWestern blots. Lane: 1, markers; 2, male SMG (60 ll extract); 3, IPR-treated male SMG treated female SMG-saliva pool (40 ll); 7, male urine (60 ll); 8, IPR-treated male urine (urine (24 h pool) (15 ll); 11, male urine (60 ll); 12, SMG-ablated male urine (60 ll); 13,Immunohistochemical localization of MSP in SMG sections: Intense cytoplasmic immunonot ductal cells of male; female shows no immunoreactivity; magnification, 200 �. (C) Wwere loaded 60 ll except SMG (2 ll) and blood-plasma fractions (2 ll). 75–100% (NH4)2

In immunohistoc hemistry of SMG sections using MSP antisera (Fig. 1B), immunoreactiv e MSP is undetect able in females while in males, it is localized within cytoplasm of acinar cells but undetectable in ductal cells. The acinar cell localization of MSP in male hamster SMG is noteworthy since reports of sex-specific proteins in salivary gland acinar cells are rare (Rosinski-Chup in et al.,1993) and to our knowledge, such abundan t sex-specific proteins are not reported in salivary glands of other mammals.

3.2. Effect of acute isoprotere nol (IPR; non-spec ific b-adrenergicagonist) treatmen t on MSP levels in SMG and its release in SMG-saliva

In rats, acute IPR treatment depletes secretory protein granules selective ly from SMG acinar cells (Iwabuchi et al., 1988; Peter et al.,1999). Interestingly, 3 h after IPR treatment of male hamsters, amarked depletion of MSP from SMG is evident [lane 3 in Fig. 1A;

P. (A) Lanes 1–10 are representative Coomassie-stained SDS–PAGE; lanes 11–14 are (60 ll); 4, female SMG (60 ll); 5, IPR-treated male SMG-saliva pool (40 ll); 6, IPR- 10 ll); 9, IPR-treated SMG-ablated male urine (10 ll); 10, Post-IPR treatment male IPR-treated male urine (0.5 ll); 14, IPR-treated SMG-ablated male urine (60 ll). (B)reactivity evidenced by golden brown DAB reaction product is present in acinar but estern blot of MSP in tissues of IPR-treated and untreated males. All tissue extracts SO4 fraction of blood-plasma is shown (see Section 2.3 and 3.4 ).


compare with lane 2 wherein equal volume (60 ll � 1/325 of SMG extracts) was loaded]. Upon comparative analysis of residual MSP bands in profiles of SMG extracts of 3 h-IPR-treated males (n = 6)with that of MSP bands in serially diluted loads of SMG extracts of untreated control males (see Section 2.6), it was estimated that acute IPR treatment of male hamsters results in >80% (86.5 ± 3.8%;mean ± SD) depletion of MSP from SMG.

In several species, including hamsters, IPR treatment elicits low volume SMG-saliva containing high protein concentr ation (Abeand Dawes, 1982 ). SMG-saliva collections (for 3 h) from control male or female (anesthetized) hamsters were negligible and 60 ll of their SMG-saliva pools (each 1.2 ml, obtained upon wash- ing the cannulat ion tubings with saline; see Section 2.4) showed novisible protein bands in Coomassie-stai ned SDS–PAGE (not shown).However, profiles of 40 ll of typical IPR-treated SMG-saliva pool (1.2 ml) of male (lane 5) and female (lane 6) shows release ofMSP in males and other proteins in both sexes. SMG of such IPR- treated males were also markedly depleted of MSP (not shown)similar to IPR-treated (conscious) males (lane 3). Since, intensity of MSP bands in lane 5 (40 ll loaded � 1/30 of SMG-saliva pool)is �1.5-fold of that in lane 2 (60 ll loaded � 1/325 of total volume of male SMG extract), it was evident that the total MSP recovered in IPR-treated male’s SMG-saliva pool comprises only a small percentage of the total MSP in a normal male’s SMG. Upon comparative analysis of Coomassie-s tained MSP bands obtained for serial dilutions of three SMG-saliva pools (of IPR-treated males) with those of SMG extracts (of normal untreated males), it was estimated that recovery of MSP in SMG-saliva pools of IPR-treated males was 16.3 ± 7.2% of total MSP in a normal male’s SMG. Thus,IPR-induced exocrine release of MSP in saliva cannot account for the >80% depletion of SMG-MSP. It needs to be mentioned here that mean total protein recovered in SMG-saliva pools of IPR-treated males was 3.5 ± 1.1 mg (n = 3), which was considerably greater than that recovered in SMG-saliva pools of untreated males (21.6 ± 4.0 lg; n = 3) (p < 0.001; Student’s t-test).

3.3. Excretion of SMG-MSP in urine of IPR-treated and normal males

Since low MSP crossreacti ons were earlier detected in Western blots of male hamster urine (Thavathiru et al., 1999 ), we investigated whether IPR treatment alters MSP levels in bladder-urine collected after 3 h (see Section 2.3). Fig. 1A shows that, while MSP-like proteins are undetect able in SDS–PAGE of 60 ll of normal male urine (lane 7), abundant 24 and 20.5 kDa MSP-like proteins are detectable in 10 ll urine of IPR-treated males (lane 8) but are undetectabl e in urine of IPR-treated-SM G-ablated males (lane 9).However, Western blots of 60 ll of normal male urine (lane 11) revealed low 24 and 20.5 kDa MSP crossreacti ons whereas 0.5 ll ofIPR-treated male urine (lane 13) showed massive MSP crossreactions and a minor �30 kDa crossreaction (another higher N-glycosylated form of MSP (Thavathi ru et al., 1999 )). Importantly, no MSP crossreactions are detectable in urine of untreated SMG-ablated males (lane 12) and IPR-treated-SM G-ablated males (lane 14). Re- sults indicate that, (i) MSP levels in normal male urine is low but enormously increases after IPR treatment, (ii) MSP in urine of normal and IPR-treated males must be sourced from SMG and itshould be routed via blood. Importan tly, the copious urinary excretion of SMG-MSP after IPR treatment, which is detectable by SDS–PAGE, is the most easily demonstrab le evidence of release into circulation of a salivary gland secretory protein, since other studies reporting release into circulation of salivary gland proteins require far intricate methods to detect them in blood (see Section 1).

Daily urine output of male hamsters of our colony is between 2–3 ml; and 0.15–0.2 ml bladder-urine is usually recoverable 3 hafter IPR treatment (see Section 2.2) whereas 0.4–0.7 ml is voided in a normal urination. While mean protein concentration of normal

(untreated) male urine was estimated to be 0.3 ± 0.1 mg/ml (n = 10), it was significantly greater for bladder urine of 3 h IPR- treated males (22.7 ± 4.1 mg/ml; n = 10; p < 0.001). Lane 10(Fig. 1A) shows SDS–PAGE of 15 ll aliquot of a typical 24 h-urine-pool (volume 2.4 ml) of an IPR-treated male maintained in ametaboli c cage after treatment. Since MSP band intensity in lane 2 (�1/325 of normal male SMG extract) is comparable to that inlane 10 (�1/160 of urine-pool), the percentage of total MSP of male SMG, which is recoverabl e in urine after IPR-treatme nt should beconsiderabl y greater than that recovered in SMG-saliva (Sec-tion 3.2). Upon comparative analysis of Coomass ie-stained MSP bands obtained for serial dilutions of four 24 h-urine-pool s (ofIPR-treat ed males) with those of SMG extracts (of untreated males), it was estimate d that the MSP recovered in the urine-pool safter IPR treatment was 52.7 ± 7.4% of total MSP in a normal (un-treated) male’s SMG. Thus, after IPR treatment, the MSP recoverable in urine is far greater than that released in SMG-saliva and both together can account to a large extent for the marked depletion of SMG-MS P. After IPR treatment, some protein bands in urine (at�50 kDa and above) usually increase in intensity. The reason for this is unclear. It needs to be mentioned here that, creatinine concentrations in urine (an indicator of kidney function) was not significantly altered 3 h after IPR treatment of hamsters.

3.4. SMG-MSP in blood and other tissues of IPR-treated and untreated males

From the above results, it can be inferred with almost certainty that a massive increase in SMG-MSP’s entry into circulation occurs after IPR-treatme nt. It is possible that after release from SMG and while in circulation, MSP can be absorbed into different tissues. Wetherefore investigated by Western blot, MSP’s presence in blood- plasma (in different (NH4)2SO4 fractions ; see Section 2.3) and se- lected tissues collected from IPR-treated and normal males.

Fig. 1C shows that 24, 20.5 (and 30) kDa MSP crossreactions asdetected for normal male SMG was also detected in 75–100%(NH4)2SO4 fraction of blood-plasma from kidney-ligated -IPR-treated males (see Section 2.3) while same fraction of blood-plasma from untreated- kidney-ligated males, showed no crossreaction.Among other fractions of blood-pla sma, only the 60–75% fraction of kidney-li gated-IPR-trea ted males, revealed low MSP crossreactions (not shown). Moreover, while kidney and submandibula rlymph nodes from untreated normal males showed no MSP crossreactions , kidney collected 3 h after IPR treatment and submandibular lymph nodes removed 45 min after IPR treatment showed MSP crossreacti ons (Fig. 1C). However, submandibula r lymph nodes removed 3 h after IPR-treat ment, showed markedly reduced crossreacti ons (not shown). Notably liver, lungs, spleen, testis and brain (which do not express MSP and show no MSP crossreaction in normal males (Thavathi ru et al., 1999 )) did not show MSP crossreaction when collected from IPR-treated males (Fig. 1C). Results strongly confirm MSP’s presence in blood of IPR-treated males and indicate no uptake of MSP in tissues, which tested negative.Submandi bular lymph node crossreacti ons suggest that some MSP released from SMG acinar cells enters interstitial spaces and is drained by lymph, which flows via these lymph nodes to re-enter circulation (Munday et al., 2005 ) (although such MSP may also directly enter blood through local capillaries ). The kidney crossreaction should represent MSP in transit within the tissue after glomerular filtration. Although, SMG of normal males releases some MSP into circulation (excreted and detectable in urine;Fig. 1A), inability to detect MSP in blood, submandibula r lymph nodes and kidney of IPR-untreated males (Fig. 1C), must be due to its extremely low concentration. However, MSP’s detection innormal male urine (Fig. 1A) was possible probably due to consider- able increase in its concentration (over blood) after substanti al


water reabsorption from glomerular filtrate, which occurs in des- ert-adapted Syrian hamster kidney having a single long papilla with large renal pelvis (Dwyer and Schmidt-N ielsen, 2003 ).

Interestingl y, after ip or sc injection of hamsters (male or female) with purified natural or recombinant MSP or even crude extract of male SMG, most of the MSP was recovered in urine (resultsnot shown). It is possible that the low mass and/or some unique feature of MSP lipocalins facilitate their entry into circulation and also unhindered urinary excretion. However , IPR-treated- SMG-ablate d males or IPR-treated females, when frequent lysprayed with a concentrated (10%) male SMG extract into their mouth and cheek-po uch and also gavaged repeatedly with the same extract, showed no detectable excretion of MSP in urine (re-sults not shown). This ruled out a possibility that, in IPR-treated males, MSP released in saliva may enter circulation upon absorp- tion across buccal, cheek-po uch or gastroint estinal mucosa.

3.5. Dose- and time-dependent depletion of SMG-MSP and its regain,after acute IPR treatment

Dose-depend ent and time-dep endent decrease in SMG-MSP levels and its time-depend ent regain after IPR treatment is depicted in Fig. 2. Males injected with 0.3 mg IPR (and sacrificed after 3 h) show �40% decrease in SMG-MSP levels, while males injected 1 or 3 mg showed >80% decrease, and 6 mg IPR was only slightly more effective (Fig. 2A). In time-depend ency studies (Fig. 2B),males injected 3 mg IPR showed no apparent decrease in SMG- MSP levels after 20 min. This could be due to either no release ofMSP or presence of most of the freshly released MSP within the SMG tissue, either in ducts or in interstiti al spaces. However, per- ceptible decrease in SMG-MSP was seen after 40 min, which de- creased further in subsequent time-points with >80% decrease observed after 3 h. Investigation of regain of SMG-MSP levels after a single 6 mg dose of IPR (Fig. 2C) showed that, at 24 h post-treatment, SMG-MSP levels remain markedly depleted (�10% of control). However, after 3 days, MSP levels increased to �25% ofcontrol and then recovered to almost control levels after 7 days.IPR treatment is known to markedly alter expression of specificgenes in salivary glands of rodents including hamsters (Mehansh oet al., 1987; Ten Hagen et al., 2002; Venkatesh et al., 2007 ). How-

Fig. 2. Dose- and time-dependent depletion of SMG-MSP and its regain, after IPR treatmeDose-dependent depletion of MSP from SMG after IPR treatment. Male hamsters were adafter 3 h; 0 mg indicates untreated control. (B) Time-dependent depletion of MSP from mhamsters and then they were sacrificed at different post-treatment times (indicated abovIPR treatment. Male hamsters were injected with IPR at a dose of 6 mg and sacrificed atuntreated control. Control males (in A, B and C) were sacrificed immediately before IPR

ever, Northern blot analysis of MSP transcript levels in SMG, collected at 1 h or 24 h after acute IPR treatment of male hamsters,revealed no difference compared to SMG of untreated males (datanot shown), which ruled out any rapid effect of IPR on MSP gene transcrip tion.

3.6. Effect of treatments with specific b-adrenergic agonists and co- treatmen ts with specific b-blockers on MSP levels in SMG and its excretion in urine

Cell-surfa ce localized b-adrenoc eptor stimulated signaling cascade in salivary glands mainly occurs through G-protein coupled adenylat e cyclase-m ediated generation of intracellular cAMP. This is followed by cAMP-depende nt activation of protein kinase-A and subsequent phosphorylatio n of specific intracellul ar proteins (Na-ter and Rohleder, 2009; Proctor and Carpenter, 2007; Ten Hagen et al., 2002 ). IPR is an agonist for both b1- and b2-adrenoceptor son salivary acinar cells. However , its binding to only b1-adrenocep-tors activates the b-adrenergic signaling cascade, which triggers exocytoti c release of secretory proteins across apical membrane into saliva (exocrine secretion) (Iwabuchi et al., 1988; Nater and Rohleder, 2009; Proctor and Carpenter , 2007 ). We therefore investigated whether, the enhanced release of MSP (from SMG) and its entry into circulation (evidenced by its copious excretion in urine),which results after IPR treatment, also occurs after treatment with specific b1- or b2-adrene rgic agonists (Abramson et al., 2003; Bedi,1993; Iwabuchi et al., 1988 ); and if so, whether co-treatment with specific b1/b2-blockers (Bedi, 1993; Iwabuchi and Masuhara, 1992;Iwabuchi et al., 1988 ) can prevent these effects. It needs mention here that the b-agonists tested, might have increased release ofMSP into saliva but this was not investigated .

Representat ive SDS–PAGE profiles of SMG and urine of differ- ently treated males are depicted in Fig. 3A and B respectivel y(wherein lane 6 represents untreated males). Treatments with IPR (lane 5), (�)-IPR (another potent non-specific b-agonist; lane 7), dobutamine (b1-agonist; lane 8) and also metaprotere nol and salbutam ol (both b2-agonis ts; lanes 10 and 13) depleted SMG- MSP and markedly increased its levels in urine. When IPR-treated males were co-treated with propanolol (non-specific b-blocker;lane 4) or metopro lol (b1-blocker; lane 3) SMG-MSP’s depletion

nt of males. Representative SDS–PAGE profiles of SMG extracts (60 ll) are shown. (A)ministered a single injection of IPR (doses indicated above the panel) and sacrificedale SMG after IPR treatment. Single injection of IPR (at 3 mg dose) was given to male e the panel); 0 min indicates untreated control. (C) Regain of MSP in male SMG after different post-treatment day intervals (indicated above the panel); 0 day indicates

treatment of experimental hamsters. Arrows indicate MSP.

Fig. 3. Effect of treatment of males with b-adrenergic agonists alone or with b-blockers on MSP levels in SMG and its excretion in urine. Lanes 1–13, in A and B, show representative SDS–PAGE of SMG extracts (60 ll) and urine (10 ll). Lanes: 1, dobutamine + butoxamine; 2, IPR + butoxamine; 3, IPR + metoprolol; 4, IPR + propanolol; 5, IPR;6, untreated control; 7, (�)-IPR; 8, dobutamine; 9, dobutamine + metoprolol; 10, metaproterenol; 11, metaproterenol + butoxamine; 12, metaproterenol + metoprolol; 13,salbutamol. See Section 3.6 for specificities of b-agonists and b-blockers and Section 2.2 for doses and treatment protocol. IPR, (±)-isoproterenol; (�)-IPR, (�)-isoproterenol.SMG and urine were collected 3 h after agonist treatment.


was largely prevented and MSP levels in urine were greatly dimin- ished. Likewise, dobutamine’s effects were prevented upon co- treatment with metopro lol (lane 9) or propanolol (not shown).However, butoxami ne (b2-blocker) was ineffective in preventing the effects of IPR (lane 2) or dobutamine (lane 1). Surprisingly ,butoxamine was also ineffective in preventing the effects of metaproterenol (b2-agonis t; lane 11) on MSP levels in SMG and urine,which were however prevented upon co-treatment with metoprolol (b1-blocker; lane 12) or propanolol (not shown). Similarly, effects of salbutamol (b2-agonist) were prevented upon co- treatment with metopro lol but not butoxamine (not shown).

Above results show that treatments with IPR and both b1- and b2-agonists release SMG-MSP into circulation (since MSP levels inurine are greatly increased). Although metoprolol (a b1-blocker)blocked the effects of IPR, b1-agonist and also b2-agonists, butoxamine (a b2-blocker) could not block the effect of b2-agonists aswell as that of b1-agonist or IPR. Thus, SMG-MSP’s release into circulation after treatment with IPR, b1- and even b2-agonists are likely mediated via stimulation of b1-adrenoceptor s (presumablypresent on hamster SMG acinar cells). In this context, it needs mention that presence of b-adrenoceptors in hamster parotid salivary gland has been demonstrat ed earlier (Mehansho et al., 1987 ).

In rats, the exocrine secretory responses of salivary acinar cells to both b1- and b2-agonists are mediated via b1-adreno ceptors (Iwabuchi et al., 1988; Nater and Rohleder, 2009 ). Moreover, inmale hamsters, exocrine secretion of proteins (and fluid) into SMG-saliva was proposed to be mediated via b1- but not b2-adrenoceptor s (Iwabuchi and Masuhara, 1992 ). Thus, b-adrenergicregulation of MSP’s release from SMG (for entry into circulation) issimilar to b-adrenergic regulation of exocrine secretion of proteins by salivary glands since both are mediated by b1-adrenocep tors.

An earlier study using disc gel electrophoresis (anionic and cat- ionic) had shown that, the types of proteins secreted in SMG-saliva of male hamsters after treatment with IPR, a-adrenergic or cholinergic agonists, were independen t of the stimulus (Abe and Dawes,1982). Therefore, since we found that IPR induces MSP’s release inSMG-saliva, it is possible that treatments with a-adrenergic orcholinergic agonists may also release MSP in SMG-saliva. This possibility, as well as the possibility that above treatments might con- comitantly release MSP into circulation, needs investigation bySDS–PAGE and/or Western-blo t of saliva and urine of treated hamsters. Moreover, aggressive behavior in mice is reported to induce release of specific proteins from SMG ductal cells into blood and saliva (Nexø et al., 1981; Rougeot et al., 2000 ). During stress (aggression-induced or otherwis e) increased release of noradrena-

line (at sympathetic nerve endings in salivary gland) and adrena- line (into blood, from adrenals) can result in adrenerg icstimulati on of SMG mediated via a- and b-adrenoc eptors (Naterand Rohleder, 2009; Proctor and Carpenter, 2007 ). Therefore, itneeds investigation whether there is any increase in release ofSMG-MS P into blood and saliva in stressed hamsters.

3.7. Possible means by which MSP may be released from SMG to enter circulatio n and significance of its presence in blood, urine and saliva

In unstimulate d normal males, continuous release of low amounts of MSP from SMG acinar cells into circulation (evidencedby low levels of MSP detected in urine) might be due to low constitutive secretion across basolateral membrane (Castle and Castle,1998; Gorr et al., 2005 ) into interstiti al spaces, from where MSP might enter circulation, directly through local capillaries or after being drained by lymph. The low MSP levels found in whole saliva (Thavathi ru et al., 1999 ) might be due to constitutive/co nstitutive -like or minor-regul ated pathway of exocrine secretion across apical membran e (Castle and Castle, 1998; Gorr et al., 2005 ).

As earlier mentioned in Section 1, certain lipocalins present insalivary glands/sal iva of different mammali an species were found to be human allergens and similar proteins were also detected inurine (Hilger et al., 2012; Torres et al., 2012 ) but the tissue source and routing of these urinary lipocalin allergens is unknown. Since our results of SMG ablation experiments indicate that urinary MSP lipocalin , in normal (unstimulated) male Syrian hamsters, issourced from SMG and routed via blood to urine, it is quite possible that some of the urinary lipocalin allergens detected in other mammalian species are also sourced from their counterpar ts in salivary glands after their release into circulation.

In IPR-stimula ted male hamsters, the markedly enhanced release of MSP from SMG acinar cells into saliva and circulation might be due to an unusual, regulated secretion occurring both apically (exocrine) and basolaterally (endocrine-like); the latter,resulting in MSP’s release into interstitium and consequent entry into circulation. Recently, it was reported that chronic IPR stimulation of rat parotid induces alkalization of acinar cell secretory granule cargo proteins (that are normally apically routed for exocrine secretion) (Venkatesh et al., 2007 ). This alkalization was proposed to be due to increased synthesis and incorporation ofbasic proteins within the secretory granules, which results intheir partial re-routing to the basolateral membrane for endocrine-like release (Gorr et al., 2005; Venkatesh et al., 2007 ). How- ever, such a mechanism cannot be responsib le for the IPR-


regulated enhanced endocrin e-like release of MSP from SMG,since it occurs within hours after acute treatment. Alternatively ,since gene transfer experime nts on murine SMG indicated that sequence features in secretory proteins can influence their sorting to apical and/or basolateral membran es (Samuni et al., 2008 ),MSP’s unusual IPR/ b-agonist-regul ated exocrine and endocrine- like secretion may be somehow influenced by its amino acid sequence.

Is it possible that no endocrin e-like release of SMG-MSP (acrossbasolateral membran es of acinar cells) occurs after b-agonist treatment? If so, then how does MSP enter circulation? One possibility is that, after b-agonist treatment intercellular tight-juncti ons inhamster SMG (between acinar or ductal cells) become leaky, which allows paracellul ar leakage of the exocrine secreted MSP, into interstitial spaces and from there MSP enters circulation (Isenmanet al., 1999; Mazariegos et al., 1984 ). However , even during copious exocrine release of amylase (an abundan t parotid acinar cell protein) into saliva induced by either direct sympatheti c nerve stimulation of rat parotid gland or acute treatment of rats with IPR (a b-sympathomi metic), no concomitan t increase in circulating levels ofamylase is detectable, indicating that no paracellular leakage occurs in rat parotid salivary gland upon b-adrenoceptor stimulati on(Isenman et al., 1999; Proctor et al., 1989 ). Moreover, freeze-frac- ture replicas of acute IPR-stimulated rat parotid gland showed noobvious gaps or discontinui ties in the intercellular tight-juncti onmeshwork (Mazariegos et al., 1984 ) and studies on SMG of chron- ically IPR-treated rats suggested that permeab ility barrier at the le- vel of acinar cells may actually become tighter (Inoue et al., 1987 ).Furthermore, the IPR-elicited volume and flow-rate of SMG-saliva is much lower in hamsters than in rats and mouse (Abe and Dawes,1982; Iwabuchi and Masuhara, 1992 ) and therefore, presumably also the pressure gradient, on which the extent of any paracellular leakage should depend (Isenman et al., 1999 ). Thus, even if any IPR-induced paracellul ar leakage (of MSP) occurs in hamster SMG, it would be expected that the ‘leaked MSP’, (which we recover in urine) would be far less than that retained and voided insaliva. Since this is not what we found, it seems that paracellular leakage is unlikely to be the reason for the massive entry ofSMG-MSP into circulation.

Interestingl y, chronic IPR/ b-agonist treatment induces several- fold increase in weight (and size) of SMG and parotid glands in rats and mice (Inoue et al., 1987; Iwabuchi et al., 1988; Mehansho et al.,1987; Selye et al., 1961; Ten Hagen et al., 2002 ) but such effects are negligible in hamsters (Mehansho et al., 1987 ) (also confirmed byus; data not shown) indicating that responses of SMG (and parotid)to IPR/ b-agonist treatment can be strikingly different between hamsters and closely related rodents. Thus, it needs investigatio n,whether other secretory mechanism (s) or a massive leakage ofSMG-MSP somehow occurs uniquely in hamster SMG in response to acute IPR/ b-agonist treatment (which results in release of MSP into circulation in far excess of exocrine release). Conversely, itneeds investigatio n whether this unique response of hamster SMG to acute IPR/ b-agonist treatment is anyway related with the reported lack of increase in SMG weight (and size) after chronic treatment.

Any role for MSP must be compatib le with its male-specificpresence in blood, urine and saliva and also its identity with odorant/pheromon e-binding lipocalin s (Srikantan et al., 2005; Thava- thiru et al., 1999 ). It is possible that MSP binds and transports pheromones /odorants for their slow and sustained release into the environment, which is required for intra-species chemical communicati on (Srikantan et al., 2005; Thavathiru et al., 1999 ).MSP’s release into blood/urine and saliva from SMG, in normal oradrenergica lly-stimulated male hamsters, might be essential for it to encounter and bind its specific ligands while circulating inblood or incubating in saliva and urine.

Abnorma l excretion of proteins in human urine has an important diagnostic value and a lipocalin , LCN2 (neutrophil gelatin- ase-associ ated lipocalin ) is an important urinary biomarker for several disease states (Soni et al., 2010 ). Although, no human coun- terpart (orthologue) of MSP lipocalin is known to be expressed inhuman salivary glands or other tissues (Srikantan et al., 2005 ),our results indicate a need to investigate whether therapeutic use of b-adrenergic agonists (for cardiac disorders , asthma etc.)(Abramson et al., 2003; Singer and Brealey, 2011 ), may release any proteins or other lipocalin s from human salivary glands (and/or other tissues) into circulation (which may be excreted and detectable in urine), since this may be a reason for side-effects of such drugs (Abramson et al., 2003; Singer and Brealey, 2011 ).

In summary , acute b-adrenergic stimulati on of hamster SMG isunique in that it induces massive release into circulation (as well as saliva) of an abundantly expressed acinar cell secretory protein,which is subsequent ly excreted in urine and easily detectab le bySDS–PAGE. This enhanced urinary excretion , is the most compelling and easily demonst rable evidence of release into circulation of a salivary gland secretory protein. The well characterized , stable and abundant MSP proteins with distinct N-glycosylated and non- glycosyla ted forms, expressed sex- and tissue-speci fically only inSMG acinar cells of male hamsters can be a model to investigate mechanis ms of release into circulation and exocrine secretion ofsalivary gland proteins.

Acknowled gments

VPD thanks CSIR (India) for research fellowship and PKD thanks DST (India) for a research grant. Prof. AM Mohr, UMDNJ-New Jersey Medical School, USA kindly gifted us butoxamine.

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copious urinary excretion of a male syrian hamster (mesocricetus auratus) salivary gland protein...

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