Altered influence of CCK-B/gastrin receptors on HDC expression in ECL cells after neoplastic transformation

Gastrin is one of the main factors controlling enterochromaffin-like (ECL) cell endocrine function and growth. Long-standing hypergastrinemia may give rise to ECL cell carcinoids in the gastric corpus in man and in experimental models. We have analysed the expression and function of CCK-B/gastrin receptors in normal ECL cells and in ECL cell tumours (gastric carcinoids) of the African rodent Mastomys natalensis. Hypergastrinemia induced by short-term (5 days) histamine2-receptor blockade (loxtidine) resulted in increased histidine decarboxylase (HDC) mRNA expression in the gastric oxyntic mucosa. This increase was significantly and dose-dependently reversed by selective CCK-B/gastrin receptor blockade (YM022). Long-term (12 months) hypergastrinemia, induced by histamine2-receptor blockade, gave rise to ECL cell carcinoids in the gastric oxyntic mucosa. CCK-B/gastrin receptor mRNA was only slightly elevated while HDC mRNA expression was eight-fold elevated in ECL cell carcinoids and was not influenced by CCK-B/gastrin receptor blockade. Thus CCK-B/gastrin receptor blockade of hypergastrinemic animals reduces the HDC mRNA expression in normal mucosa but not in ECL cell carcinoids. These results demonstrate that HDC mRNA expression in neoplastic ECL cells is not controlled by CCK-B/gastrin receptors.     

PACAP mediates the neural proliferative pathway of Mastomys enterochromaffin-like cell transformation.

BACKGROUND AND AIM: Pituitary adenylate-cyclase activating peptide (PACAP) is a more potent proliferative agent than gastrin for rat enterochromaffin-like (ECL) cell proliferation in vitro. The role of this neurotransmitter during gastrin-mediated ECL cell tumor formation and gastrin-autonomous ECL cell neoplasia is unknown. METHODS AND RESULTS: ECL cell transformation was induced in the Mastomys using 16 wk H2 receptor blockade of acid inhibition. Examination of the epithelial fundic mucosa demonstrated that PACAP-immunoreactivity significantly increased in the tumor mucosa compared to the na?ve stomach, and was associated with ECL cells. Na?ve and tumor ECL cells were then purified (approximately 95%) from Mastomys and the presence of all three PACAP/VPAC receptor subtypes was demonstrated by polymerase chain-reaction amplification. Thereafter, cells were maintained in short-term (48 h) primary cultures. PACAP significantly (p<0.05) increased 24 h bromo-deoxyuridine uptake (approximately 4-fold) in both cell types with estimated EC(50) values of approximately 4x10(-16) M and approximately 2x10(-16) M, respectively. Specific receptor antagonists (PAC1/VPAC1) of PACAP competitively inhibited these proliferative effects in na?ve cells. Oligonucleotide antisense directed against PAC1 significantly inhibited PACAP-stimulated DNA synthesis by approximately 85% (p<0.05) in tumor cells. CONCLUSION: PACAP is a potent and effective modulator of ECL cell proliferation. The expression of this neuropeptide and its receptors, particularly PAC1, suggest the existence of a neural regulatory pathway of ECL cell proliferation and transformation.     

The mechanism of histamine secretion from gastric enterochromaffin-like cells

Enterochromaffin-like (ECL) cells play a pivotal role in theperipheral regulation of gastric acid secretion as they respond to thefunctionally important gastrointestinal hormones gastrin andsomatostatin and neural mediators such as pituitary adenylate cyclase-activating peptide and galanin. Gastrin is the keystimulus of histamine release from ECL cells in vivo and in vitro.Voltage-gated K⁺ andCa²⁺ channels have been detectedon isolated ECL cells. Exocytosis of histamine following gastrinstimulation and Ca²⁺ entry acrossthe plasma membrane is catalyzed by synaptobrevin andsynaptosomal-associated protein of 25 kDa, both characterized as asoluble N-ethylmaleimide-sensitivefactor attachment protein receptor protein. Histamine release occursfrom different cellular pools: preexisting vacuolar histamineimmediately released by Ca²⁺ entryor newly synthesized histamine following induction of histidine decarboxylase (HDC) by gastrin stimulation. Histamine is synthesized bycytoplasmic HDC and accumulated in secretory vesicles byproton-histamine countertransport via the vesicular monoaminetransporter subtype 2 (VMAT-2). The promoter region of HDC containsCa²⁺-, cAMP-, and protein kinaseC-responsive elements. The gene promoter for VMAT-2, however, lacksTATA boxes but contains regulatory elements for the hormones glucagonand somatostatin. Histamine secretion from ECL cells is thereby under acomplex regulation of hormonal signals and can be targeted at severalsteps during the process of exocytosis.

     

Expression of the chromogranin A-derived peptides pancreastatin and WE14 in rat stomach ECL cells

The ECL cells constitute the predominant endocrine cell population in the mucosa of the acid-secreting part of the stomach (fundus). They are rich in chromogranin A (CGA), histamine and histidine decarboxylase (HDC). They secrete CGA-derived peptides and histamine in response to gastrin. The objective of this investigation was to examine the expression of pancreastatin (rat CGA_266-314) and WE₁₄ (rat CGA_343-356) in rat stomach ECL cells. The distribution and cellular localisation of pancreastatin- and WE₁₄-like immunoreactivities (LI) were analysed by radioimmunoassay and immunohistochemistry with antibodies against pancreastatin, WE₁₄ and HDC. The effect of food deprivation on circulating pancreastatin-LI was examined in intact rats and after gastrectomy or fundectomy. Rats received gastrin-17 (5 nmol/kg/h) by continuous intravenous infusion or omeprazole (400 μmol/kg) once daily by the oral route, to induce hypergastrinemia. CGA-derived peptides in the ECL cells were characterised by gel permeation chromatography. The expression of CGA mRNA was examined by Northern blot analysis. Among all of the endocrine cells in the body, the ECL cell population was the richest in pancreastatin-LI, containing 20–25% of the total body content. Food deprivation and/or surgical removal of the ECL cells lowered the level of pancreastatin-LI in serum by about 80%. Activation of the ECL cells by gastrin infusion or omeprazole treatment raised the serum level of pancreastatin-LI, lowered the concentrations of pancreastatin- and WE₁₄-LI in the ECL cells and increased the CGA mRNA concentration. Chromatographic analysis of the various CGA immunoreactive components in the ECL cells of normal and hypergastrinemic rats suggested that these cells respond to gastrin with a preferential release of the low-molecular-mass forms.     

CCK2 receptor antagonists: pharmacological tools to study the gastrin-ECL cell-parietal cell axis

Gastrin-recognizing CCK2 receptors are expressed in parietal cells and in so-called ECL cells in the acid-producing part of the stomach. ECL cells are endocrine/paracrine cells that produce and store histamine and chromogranin A (CGA)-derived peptides, such as pancreastatin. The ECL cells are the principal cellular transducer of the gastrin-acid signal. Activation of the CCK2 receptor results in mobilization of histamine (and pancreastatin) from the ECL cells with consequent activation of the parietal cell histamine H2 receptor. Thus, release of ECL-cell histamine is a key event in the process of gastrin-stimulated acid secretion. The oxyntic mucosal histidine decarboxylase (HDC) activity and the serum pancreastatin concentration are useful markers for the activity of the gastrin-ECL cell axis. Powerful and selective CCK2 receptor antagonits have been developed from a series of benzodiazepine compounds. These agents are useful tools to study how gastrin controls the ECL cells. Conversely, the close control of ECL cells by gastrin makes the gastrin-ECL cell axis well suited for evaluating the antagonistic potential of CCK2 receptor antagonists with the ECL-cell HDC activity as a notably sensitive and reliable parameter. The CCK2 receptor antagonists YF476, YM022, RP73870, JB93182 and AG041R were found to cause prompt inhibition of ECL-cell histamine and pancreastatin secretion and synthesis. The circulating pancreastatin concentration is raised, was lowered when the action of gastrin on the ECL cells was blocked by the CCK2 receptor antagonists. These effects were associated with inhibition of gastrin-stimulated acid secretion. In addition, sustained receptor blockade was manifested in permanently decreased oxyntic mucosal HDC activity, histamine concentration and HDC mRNA and CGA mRNA concentrations. CCK2 receptor blockade also induced hypergastrinemia, which probably reflects the impaired gastric acid secretion (no acid feedback inhibition of gastrin release). Upon withdrawal of the CCK2 receptor antagonists, their effects on the ECL cells were readily reversible. In conclusion, gastrin mobilizes histamine from the ECL cells, thereby provoking the parietal cells to secrete acid. While CCK2 receptor blockade prevents gastrin from evoking acid secretion, it is without effect on basal and vagally stimulated acid secretion. We conclude that specific and potent CCK2 receptor antagonists represent powerful tools to explore the functional significance of the ECL cells.     

Differentiation of the gastric mucosa. I. Role of histamine in control of function and integrity of oxyntic mucosa: understanding gastric physiology through disruption of targeted genes

Many physiological functions of the stomach depend on an intact mucosal integrity; function reflects structure and vice versa. Histamine in the stomach is synthesized by histidine decarboxylase (HDC), stored in enterochromaffin-like (ECL) cells, and released in response to gastrin, acting on CCK(2) receptors on the ECL cells. Mobilized ECL cell histamine stimulates histamine H(2) receptors on the parietal cells, resulting in acid secretion. The parietal cells express H(2), M(3), and CCK(2) receptors and somatostatin sst(2) receptors. This review discusses the consequences of disrupting genes that are important for ECL cell histamine release and synthesis (HDC, gastrin, and CCK(2) receptor genes) and genes that are important for "cross-talk" between H(2) receptors and other receptors on the parietal cell (CCK(2), M(3), and sst(2) receptors). Such analysis may provide insight into the functional significance of gastric histamine.     

Properties of isolated gastric enterochromaffin-like cells.

The gastric enterochromaffin-like cell (ECL) has been studied in gastric fundic glands by confocal microscopy and as a purified cell preparation by video imaging of calcium signaling and measurements of histamine release. Regulation of gastric acid secretion is largely due to alterations of histamine activation of the H2 receptor on the parietal cell and can be divided into central neural regulation, with direct actions of neuronally released mediators and into peripheral regulation by substances released from other endocrine cells. Gastric neuronal stimulation of acid secretion by alteration of ECL cell function is probably mediated by pituitary adenylate cyclase activating peptide (PACAP) receptors on the ECL cell, which activate calcium signaling and histamine release. Peripheral stimulation of acid secretion via the ECL cell is largely mediated by gastrin stimulation of calcium signaling and histamine release. Gastric neuronal inhibition of ECL cell function is probably mediated by galanin inhibition of calcium signaling, and histamine release and peripheral inhibition of ECL cell function is mainly due to somatostatin release from D cells.     

The role of endogenous gastrin in the development of enterochromaffin-like cell carcinoid tumors in Mastomys natalensis: a study with the specific gastrin receptor antagonist AG-041R.

We examined the effects of a newly synthesized gastrin receptor antagonist, AG-041R, on the growth of enterochromaffin-like (ECL) carcinoid tumors in Mastomys natalensis both in vitro and in vivo. AG-041R was as potent as the well known gastrin antagonist L365,260 in inhibiting not only the gastrin-induced release of histamine from but also histidine decarboxylase (HDC) gene expression in the ECL carcinoid tumor cells. AG-041R also inhibited gastrin-induced DNA synthesis and c-fos gene expression in the tumor cells. Furthermore, AG-041R significantly inhibited the growth of the transplanted Mastomys ECL carcinoid tumors in vivo. From these data, it is concluded that endogenous gastrin is involved in the growth of ECL carcinoid tumors in Mastomys natalensis. Moreover, AG-041R is shown to have a potential as an anti-neoplastic agent for ECL carcinoid tumor of the stomach.     

Transforming growth factor-alpha directly augments histidine decarboxylase and vesicular monoamine transporter 2 production in rat enterochromaffin-like cells

For the production and vesicle storage of histamine, Enterochromaffin-like (ECL) cells express histidine decarboxylase (HDC) and vesicular monoamine transporter 2 (VMAT2). Although HDC and VMAT2 show dynamic changes during gastric ulcer healing, the control system of their expression has not been fully investigated. In the present study, we investigated the effect of transforming growth factor-alpha (TGF-alpha) and proinflammatory cytokines on HDC and VMAT2 expression in rat ECL cells. Time course changes in the expression of TGF-alpha during the healing of acetic acid-induced ulcers were studied. EGF receptor (EGFR) expression was also examined in ECL cells, whereas the direct effects of TGF-alpha and proinflammatory cytokines on HDC and VMAT2 expression in ECL cells were investigated using in vivo and in vitro models. During the process of ulcer healing, expression of TGF-alpha mRNA was markedly augmented. Furthermore, EGFR was identified in isolated ECL cells. TGF-alpha stimulated HDC and VMAT2 mRNA expression and protein production and also increased histamine release from ECL cells. Selective EGFR tyrosine kinase inhibitor tyrphostin AG1478 almost completely inhibited HDC and VMAT2 gene expression induced by TGF-alpha in vivo and in vitro. During gastric mucosal injury, TGF-alpha was found to stimulate ECL cell functions by increasing HDC and VMAT2 expression.     

Role of gastrin in the development of gastric mucosa,ECL cells and A-like cells in newborn and young rats

Histamine-producing ECL cells and ghrelin-producing A-like cells are endocrine/paracrine cell populations in the acid-producing part of the rat stomach. While the A-like cells operate independently of gastrin, the ECL cells respond to gastrin with mobilization of histamine and chromogranin A (CGA)-derived peptides, such as pancreastatin. Gastrin is often assumed to be the driving force behind the postnatal development of the gastric mucosa in general and the ECL cells in particular. We tested this assumption by examining the oxyntic mucosa (with ECL cells and A-like cells) in developing rats under the influence of YF476, a cholecystokinin-2 (CCK(2)) receptor antagonist. The drug was administered by weekly subcutaneous injections starting at birth. The body weight gain was not affected. Weaning occurred at days 15-22 in both YF476-treated and age-matched control rats. Circulating gastrin was low at birth and reached adult levels 2 weeks after birth. During and after weaning (but not before), YF476 greatly raised the serum gastrin concentration (because of abolished acid feedback inhibition of gastrin release). The weight of the stomach was unaffected by YF476 during the first 2-3 weeks after birth. From 4 to 5 weeks of age, the weight and thickness of the gastric mucosa were lower in YF476-treated rats than in controls. Pancreastatin-immunoreactive cells (i.e. all endocrine cells in the stomach) and ghrelin-immunoreactive cells (A-like cells) were few at birth and increased gradually in number until 6-8 weeks of age (control rats). At first, YF476 did not affect the development of the pancreastatin-immunoreactive cells, but a few weeks after weaning, the cells were fewer in the YF476 rats. The ECL-cell parameters (oxyntic mucosal histamine and pancreastatin concentrations, the histidine decarboxylase (HDC) activity, the HDC mRNA levels and serum pancreastatin concentration) increased slowly until weaning in both YF476-treated and control rats. From then on, there was a further increase in the ECL-cell parameters in control rats but not in YF476 rats. The postnatal development of the ghrelin cells (i.e. the A-like cells) and of the A-like cell parameters (the oxyntic mucosal ghrelin concentration and the serum ghrelin concentrations) was not affected by YF476 at any point.We conclude that gastrin affects neither the oxyntic mucosa nor the endocrine cells before weaning. After weaning, CCK(2) receptor blockade is associated with a somewhat impaired development of the oxyntic mucosa and the ECL cells. While gastrin stimulation is of crucial importance for the onset of acid secretion during weaning and for the activation of ECL-cell histamine formation and secretion, the mucosal and ECL-cell growth at this stage is only partly gastrin-dependent. In contrast, the development of the A-like cells is independent of gastrin at all stages.     

Serum gastrin and gastric enterochromaffin-like cells during estrous cycle, pregnancy and lactation, and in response to estrogen-like agents in rats

Histamine-containing enterochromaffin-like (ECL) cells are numerous in the gastric mucosa. They operate under the control of gastrin. ECL-cell tumors (gastric carcinoids) may arise as a consequence of sustained hypergastrinemia. For reasons unknown, such tumors have a female preponderance both in laboratory animals and humans. The present study consisted of four experiments exploring the possibility that gender-related factors might affect rat ECL cells. 1) A gender difference in terms of serum gastrin concentration and oxyntic mucosal histidine decarboxylase (HDC) activity appeared in Sprague-Dawley but not Wistar rats. Ultrastructural appearance of the ECL cells did not differ between genders. 2) During the different phases of the estrous cycle, the serum gastrin concentration, HDC activity and histamine concentration did not change. 3) During pregnancy, the serum gastrin concentration was suppressed, while it was increased during lactation. The HDC activity and the histamine concentration of the oxyntic mucosa were correlated with the levels of circulating gastrin. 4) Twelve-month treatment with estrogen-like agents, dieldrin and/or toxaphene (alone or in combination) was without any effect on the ECL cells neither in male nor in female rats. In conclusion, the ECL cells are under the control of gastrin, but probably not hormones that involve in the estrous cycle and pregnancy and lactation in rats. Possible gender-related factors behind the female preponderance of ECL-cell tumors remain unknown.     

Regulation of parietal cell calcium signaling in gastric glands

The ligands interacting with enterochromaffin-like (ECL) and parietal cells and the signaling interactions between these cells were investigated in rabbit gastric glands using confocal microscopy. Intracellular calcium concentration ([Ca(2+)](i)) changes were used to monitor cellular responses. Histamine and carbachol increased [Ca(2+)](i) in parietal cells. Gastrin (1 nM) increased [Ca(2+)](i) in ECL cells and adjacent parietal cells. Only the increase of [Ca(2+)](i) in parietal cells was inhibited by H(2) receptor antagonists (H(2)RA). Gastrin (10 nM) evoked an H(2)RA-insensitive [Ca(2+)](i) increase in parietal cells. Carbachol produced large H(2)RA- and somatostatin-insensitive signals in parietal cells. Pituitary adenylate cyclase-activating peptide (PACAP, 100 nM) elevated [Ca(2+)](i) in ECL cells and adjacent parietal cells. H(2)RAs abolished the PACAP-stimulated [Ca(2+)](i) increase in adjacent parietal cells. Somatostatin did not inhibit the increase of [Ca(2+)](i) in parietal cells stimulated with histamine, high gastrin concentrations, or carbachol but abolished ECL cell calcium responses to gastrin or PACAP. Hence, rabbit parietal cells express histaminergic, muscarinic, and CCK-B receptors coupled to calcium signaling but insensitive to somatostatin, whereas rabbit and rat ECL cells express PACAP and CCK-B calcium coupled receptors sensitive to somatostatin.     

Histamine and histidine decarboxylase are hallmark features of ECL cells but not G cells in rat stomach

The oxyntic mucosa of the rat stomach is rich in ECL cells which produce and secrete histamine in response to gastrin. Histamine and the histamine-forming enzyme histidine decarboxylase (HDC) have been claimed to occur also in the gastrin-secreting G cells in the antrum. In the present study, we used a panel of five HDC antisera and one histamine antiserum to investigate whether histamine and HDC are exclusive to the ECL cells. By immunocytochemistry, we could show that the ECL cells were stained with the histamine antiserum and all five HDC antisera. The G cells, however, were not stained with the histamine antiserum, but with three of the five HDC antisera. Thus, histamine and HDC coexist in the ECL cells (oxyntic mucosa) but not in G cells (antral mucosa). Western blot analysis revealed a typical pattern of HDC-immunoreactive bands (74, 63 and 54 kDa) in oxyntic mucosa extracts with all five antisera. In antral extracts, immunoreactive bands were detected with three of the five HDC antisera (same as above); the pattern of immunoreactivity differed from that in oxyntic mucosa. Food intake of fasted rats or treatment with the proton pump inhibitor omeprazole raised the HDC activity and the HDC protein content of the oxyntic mucosa but not of the antral mucosa; the HDC activity in the antrum was barely detectable. We suggest that the HDC-like immunoreactivity in the antrum represents a cross-reaction with non-HDC proteins and conclude that histamine and HDC are hallmark features of ECL cells but not of G cells.     

PACAP stimulates gastric acid secretion in the rat by inducing histamine release

Previous studies have shown that pituitary adenylate cyclase-activating peptide (PACAP) stimulates enterochromaffin-like (ECL) cell histamine release, but its role in the regulation of gastric acid secretion is disputed. This work examines the effect of PACAP-38 on aminopyrine uptake in enriched rat parietal cells and on histamine release and acid secretion in the isolated vascularly perfused rat stomach and the role of PACAP in vagally (2-deoxyglucose) stimulated acid secretion in the awake rat. PACAP has no direct effect on the isolated parietal cell as assessed by aminopyrine uptake. PACAP induces a concentration-dependent histamine release and acid secretion in the isolated stomach, and its effect on histamine release is additive to gastrin. The histamine H2 antagonist ranitidine potently inhibits PACAP-stimulated acid secretion without affecting histamine release. Vagally stimulated acid secretion is partially inhibited by a PACAP antagonist. The results from the present study strongly suggest that PACAP plays an important role in the neurohumoral regulation of gastric acid secretion. Its effect seems to be mediated by the release of ECL cell histamine.     

Histamine metabolism of gastric carcinoids in Mastomys natalensis.

Pharmacological inhibition of gastric acid secretion and subsequent hypergastrinemia in Mastomys natalensis is an experimental model well suited for the study of gastric carcinoid formation. The genetic susceptibility of Mastomys to develop such tumors is a feature reminiscent of the situation in patients with the MEN-1 Zollinger Ellison syndrome, in whom tumor-induced hypergastrinemia, promotes the development of gastric carcinoids. Chronic hypergastrinemia, induced by the irreversible H2-receptor antagonist loxtidine will cause carcinoid formation in Mastomys already after four to six months. As in humans, gastric carcinoids in Mastomys are mainly composed of enterochromaffinlike (ECL) cells and have low malignant potential. Administration of exogenous gastrin to normal young animals increases the expression of histidine decarboxylase (HDC) mRNA in the oxyntic mucosa within 30 minutes. Endogenous hypergastrinemia, induced by short-time loxtidine treatment (three to 29 days) enhances the expression of HDC mRNA, histamine contents and ECL cell numbers in the oxyntic mucosa. Long-term loxtidine treatment (seven to 21 months) results in sustained hypergastrinemia and tumor formation. Tumor-bearing animals exhibited an increase in HDC mRNA and histamine content in the oxyntic mucosa as well as increased urinary excretion of the main histamine metabolite, tele-methylimidazole acetic acid (MeImAA). Subsequent to cessation of loxtidine treatment for two weeks, all parameters of histamine metabolism were normalized in tumor-bearing animals. These results indicate that gastric carcinoids developing during hypergastrinemia are well-differentiated neoplasms whose histamine synthesis and metabolism is regulated by plasma gastrin.     

Somatostatin, misoprostol and galanin inhibit gastrin- and PACAP-stimulated secretion of histamine and pancreastatin from ECL cells by blocking specific Ca2+ channels

The oxyntic mucosa is rich in ECL cells. They secrete histamine and chromogranin A-derived peptides, such as pancreastatin, in response to gastrin and pituitary adenylate cyclase-activating peptide (PACAP). Secretion is initiated by Ca2+ entry. While gastrin stimulates secretion by opening L-type and N-type Ca2+ channels, PACAP stimulates secretion by activating L-type and receptor-operated Ca2+ channels. Somatostatin, galanin and prostaglandin E2 (PGE2) inhibit gastrin- and PACAP-stimulated secretion from the ECL cells. In the present study, somatostatin and the PGE2 congener misoprostol inhibited gastrin- and PACAP-stimulated secretion 100%, while galanin inhibited at most 60-65%. Bay K 8644, a specific activator of L-type Ca2+ channels, stimulated ECL-cell secretion, an effect that was inhibited equally effectively by somatostatin, misoprostol and galanin (75-80% inhibition). Pretreatment with pertussis toxin, that inactivates inhibitory G-proteins, prevented all three agents from inhibiting stimulated secretion (regardless of the stimulus). Pretreatment with nifedipine (10 microM), an L-type Ca2+ channel blocker, reduced PACAP-evoked pancreastatin secretion by 50-60%, gastrin-evoked secretion by approximately 80% and abolished the response to Bay K 8644. The nifedipine-resistant response to PACAP was abolished by somatostatin and misoprostol but not by galanin. Gastrin and PACAP raised the intracellular Ca2+ concentration in a biphasic manner, believed to reflect mobilization of internal Ca2+ followed by Ca2+ entry. Somatostatin and misoprostol blocked Ca2+ entry (and histamine and pancreastatin secretion) but not mobilization of internal Ca2+. The present observations on isolated ECL cells suggest that Ca2+ entry rather than mobilization of internal Ca2+ triggers exocytosis, that gastrin and PACAP activate different (but over-lapping) Ca2+ channels, that somatostatin, misoprostol and galanin interact with inhibitory G-proteins to block Ca2+ entry via L-type Ca2+ channels, and that somatostatin and misoprostol (but not galanin) in addition block N-type and/or receptor-operated Ca2+ channels.