Relationship of fluid and mucin secretion to morphological changes in the perfused rat submandibular gland

Transport of electrolytes/water and exocytosis are activated by elevation of the cytosolic Ca(2+) concentration and are potentiated by elevation of cytosolic cyclic AMP. To correlate mucin and fluid secretion with morphological changes, rat submandibular glands were vascularly perfused and the fluid secretion and N-acetylgalactosamine in the saliva were measured during stimulation with various concentrations of carbachol (CCh) and/or isoproterenol (ISP). Single stimulation with 1 microM CCh induced a transient increase of N-acetyl galactosamine followed by a decline to a low level during sustained stimulation. The overload of 1 microM ISP increased secretion of N-acetyl galactosamine to a higher sustained level of 40-50 microg/g-min. However, at 1 microM CCh, fluid secretion was maintained at the same level during stimulation and even overload of 1 microM ISP did not significantly affect its level, whereas addition of 0.5 microM ISP to the gland stimulated with 0.1 microM CCh increased fluid secretion. Morphological observation was carried out by HRSEM and TEM. Combination of CCh and ISP in different concentrations resulted in distinctive morphological changes which reflect fluid secretion and mucin secretion. The kinetics of ATP and creatine phosphate (PCr) were measured using P-31 NMR, which indicated that the potentiation of fluid secretion is limited under a higher level of CCh stimulation due to a limited energy supply.     

Effect of hydrogen peroxide on secretory response, calcium mobilisation and caspase-3 activity in the isolated rat parotid gland

The parotid glands are highly active secretory systems subjected to continuous stress, which in turn, can lead to several pathophysiological conditions. Damage of the parotid glands are caused by radical oxygen species (ROS) as by-products of oxygen metabolism. This study investigated the effect of hydrogen peroxide (H(2)O(2)) on Carbachol (CCh)-evoked secretory responses and caspase-3 activity in the isolated rat parotid gland to understand the role of oxidative stress on the function of the gland. Amylase secretion, cytosolic calcium concentration ([Ca(2+)](i)) and caspase-3 activity in parotid gland tissue were measured using fluorimetric methods. H(2)O(2) had little or no effect on amylase secretion compared to basal level. Combining H(2)O(2) with CCh resulted in an attenuation of the CCh-evoked amylase secretion compared to the effect of CCh alone. CCh can evoke a large increase in [Ca(2+)](i) comprising an initial peak followed by a plateau. In a Ca(2+)-free medium containing 1 mM EGTA, CCh evoked only the initial peak of [Ca(2+)](i). H(2)O(2) alone evoked a gradual and dose-dependent increase in [Ca(2+)](i). Combining H(2)O(2) with CCh resulted in a decrease in [Ca(2+)](i) compared to the effect of CCh alone. In a Ca(2+)-free medium, H(2)O(2) still evoked a small increase in [Ca(2+)](i), but this response was less compared to the results obtained with H(2)O(2) in normal [Ca(2+)](0). Combining H(2)O(2) with CCh resulted in only a small transient increase in [Ca(2+)](i). Following CCh stimulation, H(2)O(2) application resulted in a large increase in [Ca(2+)](i) in normal [Ca(2+)](0). This effect of H(2)O(2) was partially abolished in a nominally free Calcium medium containing EGTA. H(2)O(2) can stimulate caspase-3 activity in parotid gland tissue. Similar response was obtained with betulinic acid and thapsigargin (TPS) on caspase-3 activity compared to basal. The results have demonstrated that like CCh, H(2)O(2) can also mobilise Ca(2+) from intracellular stores and facilitate its influx into the cell from extracellular medium. This effect of H(2)O(2) may be due to its activity to induce apoptosis in the parotid gland, since H(2)O(2) can stimulate the activity of caspase-3, a marker of cellular apoptosis.     

Relationship between cytosolic Ca2+ concentration and amylase release in rat parotid acinar cells following muscarinic stimulation.

Carbachol (CCh), a muscarinic-cholinergic agonist, increased both cytosolic free calcium concentration ([Ca2+]i) and amylase release in rat parotid acinar cells or acini in a dose-dependent manner. Treatment of acinar cells with the intracellular Ca2+ antagonist, 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8), or the intracellular Ca2+ chelator, 1,2-bis(O-aminophenoxy)ethane-N,N,N'N'-tetraacetic acid (BAPTA), strongly attenuated the increases in [Ca2+]i evoked by CCh, but amylase release from acini was not significantly suppressed by the treatment with TMB-8 or BAPTA. Low concentrations (0.02-0.5 microM) of ionomycin, a Ca2+ ionophore, caused increases in [Ca2+]i comparable to those induced by CCh, but the same concentrations had only a little effect on amylase release. The protein kinase C activator, 12-O-tetradecanoylphorbol-13-acetate (TPA), stimulated amylase release in quantities similar to those induced by CCh, although TPA alone did not cause any change in [Ca2+]i. Combined addition of TPA and ionomycin potentiated only modestly amylase release stimulated by TPA alone. Staurosporine, a protein kinase C-inhibitor, similarly inhibited both the CCh- and TPA-induced amylase release. These results suggest that an increase in [Ca2+]i elicited by CCh does not play an essential role for inducing amylase release in rat parotid acini. Amylase release by muscarinic stimulation may be mediated mainly by activation of protein kinase C.     

Stimulation of lacrimal secretion by sympathetic nerve impulses in the rabbit

Direct electrical stimulation of the preganglionic nerve trunk of the ipsilateral superior cervical sympathetic ganglion induced fluid secretion from the cannulated main excretory duct of the non-stimulated rabbit lacrimal gland. The optimum strength and frequency of stimulation were 4 volts and 25 Hz. At this stimulus parameter, the rate of secretion was 2.1 +/- 0.2 microliter/10 min. The sympathetic-induced lacrimal secretion was markedly depressed after intravenous administration of hexamethonium (1 mg/kg) and by post-ganglionic neurectomy. The results suggest that, in addition to parasympathetic nerve activity, sympathetic nerve impulses, which pass through the superior cervical sympathetic ganglion, also play a role in initiating fluid secretion in the rabbit lacrimal gland.     

Sexual differences and effects of castration on secretory mode and intracellular calcium ion dynamics of golden hamster Harderian gland

The aim of the present work was to study the sexual differences in secretory mechanisms and intracellular calcium ion dynamics in the Harderian gland of the golden hamster. In both sexes the Harderian gland consisted of small and large lobes. In the intact control male glands the secretory portions of both lobes showed wide lumina that contained secretory material and cytoplasmic fragments, suggestive of the occurrence of exocytosis and apocrine secretion. After perfusion with HEPES-buffered Ringer's solution containing 10 microM carbamylcholine (CCh), the glandular cells showed features of enhanced secretion and a rise in intracellular calcium concentration ([Ca2+]i). In the intact control female gland the lumina of most secretory portions in the large lobe contained porphyrin accretions, and exocytosis was the sole secretory mechanism. Stimulation of the large lobe with 10 microM CCh did not raise [Ca2+]i or cause enhanced secretion. The small lobe in females resembled the male gland in secretory functions, and CCh administration caused enhanced secretion and a rise in [Ca2+]i. Castration in males abolished apocrine secretion; exocytosis became the sole secretory mechanism, and stimulation of the glandular cells with CCh did not cause enhanced secretion or induce a rise in [Ca2+]i. To the contrary, in females, castration restored apocrine secretion and CCh administration caused enhanced secretion and a rise in [Ca2+]i. Castration did not affect the secretory mechanisms and the effect of CCh on the glandular cells in the small lobes of both male and female glands. The present study points to the possibility that sex hormones may control the functioning or expression of muscarinic receptors in the Harderian gland of the golden hamster.     

Octanol blocks fluid secretion by inhibition of capacitative calcium entry in rat mandibular salivary acinar cells

The aliphatic alcohol octanol is thought to modulate enzyme secretion from the exocrine pancreas by the inhibition of gap junction permeability. We have now investigated the effects of octanol on salivary secretion and intracellular calcium concentration ([Ca2+]i), measured in isolated perfused rat mandibular glands and in isolated mandibular acinar cells respectively. Stimulation of perfused glands with 10 microM carbachol (CCh) evoked a rapid increase in fluid secretion followed by a decrease to a sustained elevated level. Application of 1 mM octanol during CCh stimulation inhibited fluid secretion reversibly. In isolated acini, the CCh-induced [Ca2+]i increase was reversibly inhibited by the same concentration of octanol. However, octanol also inhibited the increase in [Ca2+]i in single acinar cells where gap junctions were no longer functional, indicating that octanol directly affected the intracellular Ca2+ signalling pathway. The initial increase in [Ca2+]i induced by 0.5-10 microM CCh, which is due to Ca2+ release from IP3-sensitive Ca2+ stores, was not affected by pretreatment with octanol. In contrast, CCh-, phenylephrine- or thapsigargin-induced Ca2+ entry was almost completely and reversibly inhibited by octanol. Octanol also blocked agonist-evoked Ca2+ entry in pancreatic acinar cells, and thapsigargin-evoked Ca2+ entry in fibroblasts. These data strongly suggest that octanol blocks salivary secretion from mandibular gland by the inhibition of capacitative Ca2+ entry, and raise the possibility that octanol may be a useful tool for inhibiting agonist-evoked Ca2+ entry pathways.     

A possible role for Ca(2+)/calmodulin-dependent protein kinase IV during pancreatic acinar stimulus-secretion coupling

Ca(2+)/calmodulin-dependent protein kinases (CaMKs) are important intracellular mediators in the mediation of stimulus-secretion coupling and excitation-contraction coupling in a wide variety of cell types. We attempted to identify and characterize the functional roles of CaMK in mediating pancreatic enzyme secretion. Immunoprecipitation and immunoblotting studies using a CaMKII or CaMKIV antibody showed that rat pancreatic acini expressed both CaMKII and CaMKIV. Phosphotransferase activities of CaMKs were measured by a radioenzyme assay (REA) using autocamtide II, peptide gamma and myosin P-light chain as substrates. Although CaMKII and CaMKIV use autocamtide II as a substrate, peptide gamma is more efficiently phosphorylated by CaMKIV than by CaMKII. Intact acini were stimulated with cholecystokinin (CCK)-8, carbachol (CCh) and the high-affinity CCK-A receptor agonist, CCK-OPE, and the cell lysates were used for REA. CCK-8, CCh and CCK-OPE caused a concentration-dependent increase in CaMKs activities. When autocamtide II was used, maximal increases were 1.5-1.8-fold over basal (20.2+/-2.0 pmol/min/mg protein), with peaks occurring at 20 min after cell stimulation. In separate studies that used peptide gamma, CCK-8, CCh and CCK-OPE dose-dependently increased CaMKIV activities. Maximal increases were 1.5-2.4-fold over basal (30.7+/-3. 2 pmol/min/mg protein) with peaks occurring at 20 min after cell stimulation. Peak increases after cell stimulation induced by peptide gamma were 1.8-2.8-fold higher than those induced by autocamtide II. CCK-8, CCh and CCK-OPE also significantly increased phosphotransferase activities of myosin light chain kinase (MLCK) substrate (basal: 4.4+/-0.7 pmol/min/mg protein). However, maximal increases induced by MLCK substrate were less than 10% of those occurring in peptide gamma. Characteristics of the phosphotransferase activity were also different between autocamtide II and peptide gamma. When autocamtide II was used, elimination of medium Ca(2+) in either cell lysates or intact cells resulted in a significant decrease in the activity, whereas it had no or little effect when peptide gamma was used. This suggests that Ca(2+) influx from the extracellular space is not fully required for CaMKIV activity and Ca(2+) is not a prerequisite for phosphotransferase activity once CaMKIV is activated by either intracellular Ca(2+) release or intracellular Ca(2+) oscillations. The specific CaMKII inhibitor KN-62 (50 microM) had no effect on the CaMKIV activity and pancreatic enzyme secretion elicited by CCK-8, CCh and CCK-OPE. The specific MLCK inhibitor, ML-9 (10 microM), also did not inhibit CCK-8-stimulated pancreatic amylase secretion. In contrast, wide spectrum CaMK inhibitors, K-252a (1 microM) and KT5926 (3 microM), significantly inhibited CaMKIV activities and enzyme secretion evoked by secretagogues. Thus, CaMKIV appears to be an important intracellular mediator during stimulus-secretion coupling of rat pancreatic acinar cells.     

Effect of beta-antagonists on isoprenaline-induced secretion of fluid, amylase and protein by the parotid gland of the red kangaroo, Macropus rufus

Selective and non-selective beta-adrenoceptor antagonists were used to block the increases in fluid, protein and amylase secretion caused by sympathomimetic stimulation of the parotid gland of red kangaroos during intracarotid infusion of isoprenaline. ICI118551 at antagonist/agonist ratios up to 300:1 caused increasing but incomplete blockade of fluid secretion, and protein/amylase release. Atenolol at antagonist/agonist ratios up to 300:1 was only marginally more potent than ICI118551 at blocking the fluid, protein and amylase responses. Propranolol at antagonist/agonist ratios of 30:1 was as effective at blocking fluid and protein secretion as the highest ratios of either atenolol or ICI118551. Simultaneous administration of atenolol (30:1) with ICI118551 (30:1) was not as potent as propranolol (30:1). Thus, the beta-adrenoceptor/s in the acini of the kangaroo parotid gland appear to have antagonist-binding affinities atypical of those found for eutherian tissues. The data are consistent with the gland possessing either a single anomalous beta-adrenoceptor or functional beta(2)-receptors in addition to the beta(1)-receptors which are characteristic of eutherian salivary glands.     

Calcium antagonists cause dry mouth by inhibiting resting saliva secretion

Ca2+ antagonists cause dry mouth by inhibiting saliva secretion. The present study was undertaken to elucidate the mechanism by which Ca2+ antagonists cause dry mouth. Since the intracellular Ca2+ concentration ([Ca2+]i) is closely related to saliva secretion, [Ca2+]i was measured with a video-imaging analysis system by using human submandibular gland (HSG) cells as the material. The Ca2+ antagonist, nifedipine, inhibited the elevation in [Ca2+]i induced by 1-10 microM carbachol (CCh), but had no inhibitory effect on that induced by 30 and 100 microM CCh. The other kinds of Ca2+ antagonists, verapamil (10 microM), diltiazem (10 microM), and the inorganic Ca2+ channel blocker, CdCl2 (50 microM), also inhibited the [Ca2+]i elevation induced by 10 microM CCh. The Ca2+ channel activator, Bay K 8644 (5 microM), significantly enhanced the CCh (10 microM)-induced [Ca2+]i elevation. Endothelin-1 and norepinephrine also increased the CCh (10 microM)-induced [Ca2+]i elevation. SKF-96365 reversed the enhancement of the CCh (10 microM)-induced [Ca2+]i elevation caused by AlF4- and phenylephrine. The phospholipase Cbeta (PLCbeta) inhibitor, U-73122 (5 microM), significantly inhibited the [Ca2+]i elevation induced by 100 microM CCh compared with that induced by 10 microM CCh, while the PLCbeta activator, m-3M3FBS (20 microM), significantly increased the [Ca2+]i elevation induced by 100 microM CCh compared with that induced by 10 microM CCh. We therefore conclude that non-selective cation and voltage-dependent Ca2+ channels are involved in resting salivation and that Ca2+ antagonists depress H2O secretion by blocking the Ca2+ channels and thereby cause dry mouth.     

Nitric oxide modulates salivary amylase and fluid, but not epidermal growth factor secretion in conscious rats

The involvement of the L-arginine/NO pathway in the control of salivary fluid, amylase and epidermal growth factor (EGF) secretion was investigated in conscious rats. For the collection of saliva, an oesophageal cannula was implanted. To obtain steady secretion, submaximal carbachol background infusion was given. Different treatments included NO synthase inhibitor N(G)-nitro-L-arginine (NOLA; with or without phentolamine, propranolol), L-arginine, D-arginine and NO donor 3-morpholinosydnonimine (SIN-1) administration. Volume, amylase activity and EGF output in the secreted fluid were determined in 30 min mixed saliva samples. Carbachol infusion alone produced a modest, sustained salivary fluid and amylase secretion. NOLA (30 mg/kg) further increased both fluid (p<0.001) and amylase outputs (p<0.001). These latter effects were prevented by L-arginine but not by D-arginine or by phentolamine. Propranolol administration decreased both fluid and amylase secretion below the carbachol plateau, and NOLA did not modify this suppressed secretory rate. SIN-1 did not alter either volume or amylase secretion. Interestingly, NOLA given without carbachol did not modify salivation. Neither carbachol nor NOLA changed salivary EGF output. The present results suggest that the L-arginine/NO pathway has a modulatory role in the cholinergic control of salivary amylase secretion, but not in EGF output. The mechanisms of inhibitory action of NO on salivary fluid and amylase secretion remain to be identified.     

Secretagogue response of goblet cells and columnar cells in human colonic crypts

Crypts of Lieberkühn were isolated from human colon, and differential interference contrast microscopy distinguished goblet and columnar cells. Activation with carbachol (CCh, 100 microM) or histamine (10 microM) released contents from goblet granules. Stimulation with prostaglandin E(2) (PGE(2), 5 microM) or adenosine (10 microM) did not release goblet granules but caused the apical margin of columnar cells to recede. Goblet volume was lost during stimulation with CCh or histamine ( approximately 160 fl/cell), but not with PGE(2) or adenosine. Three-quarters of goblet cells were responsive to CCh but released only 30% of goblet volume. Half-time for goblet volume release was 3.7 min. PGE(2) stimulated a prolonged fluid secretion that attained a rate of approximately 350 pl/min. Columnar cells lost approximately 50% of apical volume during maximal PGE(2) stimulation, with a half-time of 3.3 min. In crypts from individuals with ulcerative colitis, goblet cells were hypersensitive to CCh for release of goblet volume. These results support separate regulation for mucus secretions from goblet cells and from columnar cells, with control mechanisms restricting total release of mucus stores.     

Heat production, blood flow, O2 uptake and CO2 output in the secretary process of the dog submandibular gland (author's transl)

1. Under normal circulation of the dog submandibular gland, the electrical stimulation induced a massive salivary secretion (about 0.35 ml . min-1.g-1 gland weight) and an increase in the glandular temperature (about 0.2 degrees C). The heat production was calculated of about 60 mW.g-1. 2. Clamping of the glandular artery made both of secretion and heat production to be transient. The early peak of secretion was about 0.12 ml.min-1.g and that of heat production was 7 approximately 10mW,g-1. Then each 1 ml secretion followed about 4.6 J heat production. 3. Under constant blood flow in the glandular circulation, the secretory process was divided clearly into 2 phases of peak and plateau. The glandular temperature increased about 0.12 degrees C with an early temperature drop. In the secretory plateau phase, the secretary rate was about 0.043 ml.min-1.g-1, the heat production was about 5 approximately 7 mW.g-1 and each 1 ml secretion caused about 8.2 J heat production. 4. The rate of oxygen uptake was about 20.9 microl.min-1g-1 at the resting state. The maximum during secretion was about 192 microliter.min-1.g-1. THe half time of the recovery process of O2 uptake tended slightly longer than that of heat production. 5. THe rate of CO2 output was about 21.8 microliter.min-1.g-1 at resting. The maximum during secretion was about 142 microliter.min-1.g-1 R. Q. were about 1 at resting and about 0.74 under secretion.     

Effect of thapsigargin on cytosolic Ca2+ level and amylase release in rat parotid acinar cells.

At concentrations greater than 0.01 microM, thapsigargin (ThG) dose-dependently caused an increase in cytosolic free Ca2+ concentration ([Ca2+]i) in rat parotid acinar cells, as measured by the fluorescent Ca(2+)-indicator fura-2. In the absence of extracellular Ca2+, a transient increase in [Ca2+]i by ThG was observed, and subsequent addition of carbachol (CCh) did not produce a further [Ca2+]i response, suggesting that ThG released Ca2+ from the CCh-sensitive intracellular Ca2+ pool. Since ThG did not stimulate formation of inositol phosphates, the ThG-induced Ca2+ mobilization is independent of phosphoinositide breakdown. High concentrations (greater than 0.1 microM) of ThG induced amylase release from rat parotide acini, but the effect was very poor as compared with that of CCh or the protein kinase C activator, PMA (phorbol 12-myristate 13-acetate). Combined addition of ThG and PMA modestly potentiated amylase release induced by PMA alone. These results support the view that amylase release by muscarinic stimulation is mediated mainly by activation of protein kinase C rather than a rise in [Ca2+]i, although Ca2+ may modulate the secretory response.     

The effect of L-arginine/nitric oxide pathway on salivary amylase secretion in conscious rats

In a recent study we have demonstrated the presence of nitric oxide synthase immunoreactive neurons and also perivascular, periacinar and periductal nerve fibres in feline submandibular salivary gland. The role of nitric oxide (NO) in salivary vasoregulation has been suggested by other data too, but the effect of NO on salivary amylase secretion has not been investigated yet. Under ether anaesthesia a catheter was introduced into the oesophagus for salivary juice collections, and a cannula was inserted into the jugular vein for infusions. After postanaesthesia recovery, submaximal carbachol infusion was given as a background to obtain steady secretion because of the low basal secretory rate. Then different groups of animals received NO synthase inhibitor NOLA (N^G-nitro-L-arginine), L-arginine, D-arginine or NO donor SIN-1 (3-morpholinosydnonimine). Volume and amylase activity were determined in mixed saliva samples collected for 30 min. Carbachol background infusion alone induced an elevated, sustained salivary secretion. NOLA (30 mg/kg) increased both volume and amylase output (P < 0.001). This effect was prevented by L-arginine but not by D-arginine. SIN-1 did not change either volume or amylase secretion. The present results suggest that the L-arginine/NO pathway has a modulatory effect on salivary fluid and amylase secretion, which is probably not related to its effect on salivary blood flow. NO may block certain presently unidentified secretagogue mechanisms and/or may relax myoepithelial cells.     

Effects of combined secretagogues and extracellular calcium on neonatal insulin release

The insulin response of 3-day old neonatal rat islets was evaluated following a 1 h incubation with glucose alone and in the presence of 30 nM sulfated cholecystokinin octapeptide (CCK) and/or 20 microM carbachol (CCh). Insulin secretion was found to be incrementally increased from the lowest glucose concentration and enhanced several fold in the presence of CCK and/or CCh. In combination, CCK and CCh increased glucose-stimulated insulin secretion by an amount equivalent to the sum of their individual increases. The presence of either CCK alone or CCK plus CCh increased phosphoinositide hydrolysis by the same relative amount that they increased insulin secretion when compared to 8.3 mM glucose. Glucose-stimulated insulin secretion was totally inhibited when calcium was omitted from the incubation buffer; this effect was partially negated by CCK alone and more so by CCK combined with CCh. Insulin secretion in response to 8.3 mM glucose alone was unchanged when calcium in the incubation buffer was increased from 1 to 5 mM; however, the insulin response to 16.7 mM glucose alone and 8.3 mM glucose in the presence of CCK and/or CCh was increased under this condition. Thus, we have shown that, even at 3 days postpartum, insulin secretion from isolated islets is a complex response capable of being molded by several secretagogues at once and ultimately determined by interplay of different signaling systems activated.     

Heterogeneity and contact-dependent regulation of amylase release by individual acinar cells

We have used a reverse hemolytic plaque assay to investigate the amylase release of single and aggregated pancreatic acinar cells. We have found that a minority of single acinar cells released detectable amounts of amylase under basal conditions and were modestly stimulated, in a dose-dependent manner, during a 30-min exposure to concentrations of carbamylcholine (CCh) ranging from 10^?8 to 10^?5 M. This stimulation was largely accounted for by the recruitment of additional secreting cells, rather than by a significant increase in their individual secretory output. We have also observed that aggregates comprising two to five acinar cells secreted more frequently and released more amylase than single acinar cells in the presence of each of the CCh concentrations tested. Under both basal conditions and following CCh stimulation, the proportion of secreting aggregates and their amylase output increased linearly with the aggregate size. Under basal conditions as well as in the presence of secretagogue concentrations in the 10^?8?10^?7 M range, individual cells contributed similarly to amylase secretion whether they were single or part of aggregates. By contrast, following stimulation by 10^?6?10^?5 M CCh, aggregated cells showed a much higher average secretion than single cells. Investigating the mechanism of this contact-dependent effect, we found that 10^?3 M heptanol did not significantly modify the secretion of single cells and markedly promoted the basal amylase release of acinar cell pairs. This effect was associated with a marked reduction in gap junctional communication between acinar cells, as evaluated by microinjection of Lucifer yellow, and was not observed during exposure to high concentrations of CCh, which also reduced junctional communication. These data show that pancreatic acinar cells are intrinsically heterogeneous in their ability to release amylase and that their basal as well as stimulated secretion are promoted by the establishment of direct intercellular contacts. Our experiments also suggest that junctional coupling contributes to the contact-dependent mechanism which enhances the recruitment of secreting cells and their individual output. These observations strengthen the view that direct interactions between acinar cells are essential in the control of pancreatic secretion. © 1994 Wiley-Liss, Inc.     

Salivary secretion induced by substance P

Secretion of fluid, ions, and amylase from parotid and submaxillary glands of rat, induced by intravenous injection of substance P (SP), was examined. The action of SP on salivary glands, like physalaemin, resembled that of cholinergic stimulation. While SP-evoked salivary flow from both glands was blocked by atropine, atropine did not modify composition of SP-evoked saliva. The present study suggests that salivary secretion and secretion of ions and amylase evoked by SP are mediated via SP-sensitive cholinergic receptors and specific SP receptors, respectively.     

Nonparallel secretion of enzymes by the rabbit pancreas

Since nonparallel secretion of enzymes by the exocrine pancreas has been demonstrated with several experimental models, we were interested in verifying a recent claim that enzyme secretion remained strictly proportional (parallel) upon stimulation of the in vivo rabbit pancreas. Pancreatic juice was collected by extraduodenal cannulation of the pancreatic duct, in two different protocols. In the first protocol the administration of pentobarbital induces a mild anesthesia. Under this condition, amylase and chymotrypsin secretion remained parallel after cholecystokinin stimulation. In a second protocol, a deeper and constant anesthesia was attained with Fluothane resulting in a lower basal protein output than in the first protocol. Pancreatic secretion was collected under intravenous secretin perfusion (4.5 clinical units X kg-1 X h-1). After stabilization and basal collection periods, pancreatic secretion was stimulated with an i.v. bolus injection of either cholecystokinin (2 Ivy dog units/kg), caerulein (0.1 micrograms/kg), or carbachol (6 micrograms/kg). Upon stimulation of the pancreas, protein output increased an average of 30-fold and there was a concomitant 20-25% decrease in the ratio of the specific activities of amylase to chymotrypsin which resulted from a greater increase in the specific activity of chymotrypsin in pancreatic juice after stimulation of secretion. Thus, under appropriate conditions, nonparallel secretion of enzymes by the exocrine pancreas can be demonstrated in yet another experimental model. Furthermore, the proportion of amylase and chymotrypsin activities in pancreatic juice are once more shown to be dependent, up to a threshold, upon the rate of protein output by this exocrine gland.     

Effects of an inhibitor of myosin light chain kinase on amylase secretion from rat pancreatic acini

Ca(2+)/calmodulin-dependent protein (CaM) kinases play an important role in Ca(2+)-mediated secretory mechanisms. Previously, we demonstrated that a CaM kinase II inhibitor KN-62 had a small inhibitory effect on amylase secretion stimulated by CCK. In the present study, we investigated the effects of a myosin light chain kinase (MLCK) inhibitor on amylase secretion and Ca(2+) signaling in rat pancreatic acini. A specific inhibitor of MLCK, wortmannin, inhibited amylase secretion stimulated by CCK-8 (30 pM) in a concentration-dependent manner. Wortmannin (10 microM) had no effects on basal secretion but reduced amylase secretion stimulated by CCK-8 (30 pM) by 67 +/- 3%. Wortmannin inhibited amylase secretion stimulated by calcium ionophore (A23187) and phorbol ester (TPA). Wortmannin also inhibited amylase response to thapsigargin by 76 +/- 8% and to both thapsigargin and TPA by 52 +/- 10%. Ca(2+) oscillations evoked by CCK-8 (10 pM) were inhibited by wortmannin (10 microM). Wortmannin had a little inhibitory effect on an initial rise in [Ca(2+)](i), and abolished a subsequent sustained elevation of [Ca(2+)](i) evoked by 1 nM CCK-8. In conclusion, MLCK plays a crucial role in amylase secretion from pancreatic acini and regulates Ca(2+) entry from the extracellular space.     

Changes in parotid acinar cells accompanying salivary secretion in rats on sympathetic or parasympathetic nerve stimulation

Morphological and secretory effects of stimulating autonomic nerves have been studied in parotid glands of rats. Sympathetic stimulation evoked a slow flow of saliva which had a high concentration of amylase. After long term sympathetic stimulation secretory granules were heavily depleted from the parotid acinar cells. Parasympathetic stimulation evoked a copious flow of saliva with a low concentration of amylase. However, at high frequency stimulation the total amount of amylase secreted on parasympathetic stimulation was as great or even greater than on symphatetic stimulation, nevertheless, any loss of secretory granules from the acinar cells was very small. It is concluded that secretion of parotid acinar granules in the rat is prinicipally a sympathetic function. Secretion of fluid is more effectively produced by parasympathetic stimulation and much of the amylase in such saliva appears to have arisen from sources other than the secretory granules.