Regulation of the Na+/Ca2+ exchanger in rat pancreatic ducts.

The Ca2+ content of pancreatic juice is closely regulated by yet unknown mechanisms. One aim of the present study was to find whether rat pancreatic ducts have a Na+/Ca2+ exchanger, as found in some Ca2+ transporting epithelia. Another aim was to establish whether the exchanger is regulated by hormones/agonists affecting pancreatic secretion. Whole pancreas, pure pancreatic acini and ducts were obtained from rats and used for RT-PCR and Western blot analysis, immunohistochemistry and intracellular Ca2+ measurements using Fura-2. RT-PCR analysis indicated Na+/Ca2+-exchanger isoforms NCX1.3 and NCX1.7 in acini and pancreas. Western blot with NCX1 antibody identified bands of 70, 120 and 150 kDa in isolated ducts, acini and pancreas. Immunofluorescence experiments showed the Na+/Ca2+ exchanger on the basolateral membrane of acini and small intercalated/intralobular ducts, but in larger intralobular/extralobular ducts the exchanger was predominantly on the luminal membrane. Na+/Ca2+ exchange in ducts was monitored by changes in intracellular Ca2+ activity upon reversal of the Na+ gradient. Secretin (1 nM) and carbachol (1 mM) reduced Na+/Ca2+ exchange by 40% and 51%, respectively. Insulin (1 nM) increased Na+/Ca2+ exchange by 230% within 5 min. The present study shows that pancreatic ducts express the Na+/Ca2+ exchanger. Its distinct localization along the ductal tree and regulation by secretin, carbachol and insulin indicate that ducts might be involved in regulation of Ca2+ concentrations in pancreatic juice.

cGMP-dependent and -independent inhibition of a K+ conductance by natriuretic peptides: molecular and functional studies in human proximal tubule cells.

In immortalized human kidney epithelial (IHKE-1) cells derived from proximal tubules, two natriuretic peptide receptors (NPR) were identified. In addition to NPR-A, which is bound by atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and urodilatin (URO), a novel form of NPR-B that might be bound by C-type natriuretic peptide (CNP) was identified using PCR. This novel splice variant of NPR-B (NPR-Bi) was also found in human kidney. Whereas ANP, BNP, and URO increased intracellular cGMP levels in IHKE-1 cells in a concentration-dependent manner, CNP had no effect on cGMP levels. To determine the physiologic responses to these agonists in IHKE-1 cells, the membrane voltage (Vm) was monitored using the slow whole-cell patch-clamp technique. ANP (10 nM), BNP (10 nM), and URO (16 nM) depolarized these cells by 3 to 4 mV (n = 47, 7, and 16, respectively), an effect that could be mimicked by 0.1 mM 8-Br-cGMP (n = 15). The effects of ANP and 8-Br-cGMP were not additive (n = 4). CNP (10 nM) also depolarized these cells, by 3+/-1 mV (n = 28), despite the absence of an increase in cellular cGMP levels, indicating a cGMP-independent mechanism. In the presence of CNP, 8-Br-cGMP further depolarized Vm significantly, by 1.6+/-0.3 mV (n = 5). The depolarizations by ANP were completely abolished in the presence of Ba2+ (1 mM, n = 4) and thus can be related to inhibition of a K+ conductance in the luminal membrane of IHKE-1 cells. The depolarizations attributable to CNP were completely blocked when genistein (10 microM, n = 6), an inhibitor of tyrosine kinases, was present. These findings indicate that natriuretic peptides regulate electrogenic transport processes via cGMP-dependent and -independent pathways that influence the Vm of IHKE-1 cells.

Guanylin stimulates regulated secretion from human neuroendocrine pancreatic cells.

BACKGROUND & AIMS: Gastroenteropancreatic neuroendocrine cells secrete chemical messengers in a calcium-dependent fashion. So far, other second messenger systems involved in regulated secretion have gained little attention. The aim of this study was to characterize guanosine 3',5'-cyclic monophosphate (cGMP)-mediated vesicular secretion in pancreatic neuroendocrine cells. METHODS: In a human pancreatic cell line, BON, cyclic nucleotide levels and chromogranin A release were monitored with specific immunoassays. Uptake and release of gamma-aminobutyric acid were measured. Intracellular Ca2+ concentration was monitored with fura-2. Guanylyl cyclase C was analyzed by reverse-transcription polymerase chain reaction. RESULTS: Guanylin increased cGMP concentrations in BON cells via guanylyl cyclase C. Stimulation of the cGMP pathway by guanylin or Escherichia coli heat-stable enterotoxin increased the release of chromogranin A and gamma-aminobutyric acid from BON cells. This effect was mimicked by the cGMP analogue 8-bromo-cGMP. CONCLUSIONS: Guanylin and STa stimulate the regulated secretion from BON cells via guanylyl cyclase C and cGMP. Our study yields novel information about secretory properties of guanylin, mediated via a signal transduction pathway, increasing cGMP and leading to regulated secretion of neuroendocrine cells.

Moxonidine inhibits Na+/H+ exchange in proximal tubule cells and cortical collecting duct.

The imidazoline receptor agonist moxonidine has been recently introduced as an antihypertensive therapy. Imidazoline specific binding sites have also been found in the kidney. Moxonidine induced natriuresis and diuresis in clearance studies in rats. Related substances such as various guanidinium derivatives have been shown to inhibit Na+/H+ exchange in several preparations. We therefore examined whether the renal effects of moxonidine could be mediated by an inhibition of the Na+/H+ exchanger. Intracellular pH (pHi) was measured microfluorimetrically with BCECF in proximal LLC-PK1 cells and in the principal cells of rat cortical collecting ducts (CCD). In LLC-PK1 cells moxonidine (10 mumol/liter) had no effect on the basal pH1; however, it reduced the Na+/H+ activity reversibly by 43 +/- 4% (N = 26) when the exchanger was activated by cellular acidification. In rat CCD cells moxonidine slightly decreased basal pHi by 0.08 +/- 0.03 pH units (N = 12). After acidification the recovery rate of pHi was reduced with moxonidine by 45 +/- 6% (N = 18). The effects of moxonidine could be mimicked in both cell types by inhibitors of the Na+/H+ exchanger (HOE 694, amiloride). In the presence of the imidazoline receptor antagonist idazoxan (10 mumol/liter) the effects of moxonidine were almost completely inhibited. The alpha 2-antagonist yohimbine (10 mumol/liter) did not significantly alter the effects of moxonidine in both cell types. These data suggest that in LLC-PK1 and in rat CCD cells, Na+/H+ is inhibited by moxonidine via an activation of the imidazoline receptor.

Functional evidence for the regulation of cytosolic Ca2+ activity via V1A-receptors and beta-adrenoceptors in rat CCD.

In freshly isolated rat CCD segments, the effects of arginine vasopressin (AVP), oxytocin (OT), adrenaline (Ad), and their specific receptor agonists and antagonists on the intracellular Ca2+ activity ([Ca2+]i) were measured using the Ca2+ sensitive dye Fura-2 as fluorescence indicator. We observed that AVP, the V1-receptor agonist [Phe2Orn8] vasotocin ([Phe2]OVT), and OT increased [Ca2+]i biphasically. AVP (n = 9) and OT (n = 8) induced increases in [Ca2+]i were completely blocked by the V1A-receptor antagonist d(CH2)5Tyr(Me)2AVP. However, neither the V2-receptor agonist [Val4-D-Arg8]AVP (100 nM, n = 5), nor the OT-receptor agonist [Thr4,Gly7]OT (100 nM, n = 5) nor forskolin (1 microM, n = 4 and 10 microM, n = 5) did significantly change [Ca2+]i. Ad and the beta-adrenoceptor agonist isoproterenol (ISO) increased [Ca2+]i, which was not mimicked by the alpha 2-adrenoceptor agonist clonidine (1 microM, n = 10) or the alpha 1-adrenoceptor agonist phenylephrine (1 microM, n = 5). The beta-adrenoceptor antagonist propranolol (1 microM) completely blocked this Ad (1 microM, n = 4) induced [Ca2+]i increase. Insulin (INS 10 nM, n = 8), endothelin (ET 1 microM, n = 6), and angiotensin II (Ang II 1 pM to 10 nM; each n = 4) had no significant effect on [Ca2+]i. Considering the present results we propose a V1A-receptor and beta-adrenoceptor dependent modulation of [Ca2+]i in rat CCD.

Receptors for bradykinin and prostaglandin E2 coupled to Ca2+ signalling in rat cortical collecting duct.

In freshly isolated rat cortical collecting ducts (CCD) we measured intracellular Ca2+ activity ([Ca2+]j) with the Fura-2 method. Bradykinin (BK) induced a transient and biphasic increase in [Ca2+]j. This increase was concentration dependent and was half maximal at a concentration of 15 nM. The B2 receptor antagonist HOE 140 (100 nM, n = 6) completely abolished BK (100 nM) induced increase in [Ca2+]j. The B1 receptor agonist des-Arg9-bradykinin (100 nM, n = 4) had no effect on [Ca2+]j. In the absence of extracellular Ca2+, the maximal increase in [Ca2+]j, induced by BK was diminished and the secondary plateau phase was completely abolished. Prostaglandin E2 (PGE2) elevated [Ca2+]j, also concentration-dependently and biphasically. A half maximal effect was reached with 1 nM PGE2. The secondary plateau phase was absent when extracellular Ca2+ was removed. Sulprostone (100 nM, n = 6) mimicked the PGE2 (100 nM) induced increase in [Ca2+]j. The effect of BK (100 nM) on [Ca2+]j was not inhibited by the cyclooxygenase inhibitor indomethacin (10 microM, n = 5). Dopamine (1 microM, n = 4) did not significantly alter [Ca2+]j. BK and PGE2 regulate [Ca2+]j in the rat CCD via release of Ca2+ from intracellular Ca2+ stores as well as via Ca2+ influx from extracellular space. BK directly modulates [Ca2+]j, through B2 receptors. EP1 receptors are most likely to be responsible for the PGE2 induced increase in [Ca2+]j.