Kazuro Yaekura

Pituitary adenylate cyclase-activating polypeptide (PACAP) is an islet substance serving as an intra-islet amplifier of glucose-induced insulin secretion in rats.

1 We examined whether pituitary adenylate cyclase‐activating polypeptide with 38 or 27 residues (PACAP‐38 or PACAP‐27) serves as an intra‐islet regulator of glucose‐induced insulin secretion in rats. PACAP antiserum specific for PACAP‐38 and PACAP‐27 was used to neutralize the effect of endogenous PACAP in islets. PACAP release from islets was bioassayed using the response of cytosolic Ca2+ concentration ([Ca2+]i) in single β‐cells, monitored by dual‐wavelength fura‐2 microfluorometry. Expression of PACAP mRNA was studied by reverse transcription‐polymerase chain reaction (RT‐PCR), while expression of PACAP was studied by metabolic labelling and immunoblotting. Localization of PACAP receptors was studied immunohistochemically. 2 High glucose‐stimulated insulin release from isolated islets was attenuated by PACAP anti‐serum but not by non‐immune sera. 3 The islet incubation medium with high glucose (Med) possessed a capacity, which was neutralized by PACAP antiserum, to increase [Ca2+]i in β‐cells. PACAP antiserum also neutralized the [Ca2+]iincreasing action of synthetic PACAP‐38 and PACAP‐27, but not that of vasoactive intestinal polypeptide (VIP) and glucagon. 4 Both Med and synthetic PACAP increased [Ca2+]i in β‐cells only in the presence of stimulatory, but not basal, glucose concentrations. In contrast, ATP, a substance that is known to be released from β‐cells, increased [Ca2+]i in β‐cells at both basal and stimulatory glucose concentrations. 5 Expression of PACAP mRNA and biosynthesis of PACAP‐38 were detected in islets and a β‐cell line, MIN6. 6 Immunoreactivity for PACAP‐selective type‐I receptor was observed in islets. 7 [Ca2+]i measurements combined with immunocytochemistry with insulin antiserum revealed a substantial population of glucose‐unresponsive β‐cells, many of which were recruited by PACAP‐38 into [Ca2+]i responses. 8 These results indicate that PACAP‐38 is a novel islet substance that is synthesized and released by islet cells and then, in an autocrine and/or paracrine manner, potentiates and arouses β‐cells responses to glucose, thereby amplifying glucose‐induced insulin secretion in islets.

cAMP-Signaling Pathway Acts in Selective Synergism With Glucose or Tolbutamide to Increase Cytosolic Ca2+ in Rat Pancreatic β-Cells

cAMP and the insulinotropic peptides that raise cAMP glucose-dependently increase the cytosolic free Ca2+ concentration ([Ca2+]i) in pancreatic β-cells, which is tightly linked to the potentiation of glucose-induced insulin release. We examined whether cAMP increases [Ca2+]i in specific cooperation only with glucose or also with other insulin secretagogues that act through different mechanisms. [Ca2+]i in single rat pancreatic β-cells was measured by dual-wavelength fura-2 microfluorometry. In the presence of a stimulatory concentration of glucose (8.3 mmol/l) and the moderate elevation in [Ca2+]i induced by it, forskolin, an activator of adenylyl cyclase, or dibutyryl cAMP produced a marked additional increase in [Ca2+]i but was ineffective at the basal 2.8 mmol/l glucose. These cAMP-elevating agents also potentiated the effect of tolbutamide on [Ca2+]i. The cAMP-induced increase in [Ca2+]i was completely and selectively inhibited by a blocker of cAMP-dependent protein kinase A (PKA), and by nitrendipine, a blocker of the L-type Ca2+ channel. However, in the presence of high KCl and the [Ca2+]i elevation induced by it, a rise in cAMP failed to further increase [Ca2+]i, whereas BAY K8644, an agonist of L-type Ca2+ channels, evoked an additional increase in [Ca2+]i. Under low Na+ conditions, the [Ca2+]i response to cAMP was observed in the majority of the cells. In the cells in which glucose at 4.5–5 mmol/l was inadequate to increase [Ca2+]i, the glucose together with a rise in cAMP often increased [Ca2+]i. Likewise, tolbutamide and a rise in cAMP acted in concert to increase [Ca2+]i. Thus, cAMP left-shifted the concentration-[Ca2+]i response relationship for glucose and tolbutamide. In conclusion, the cAMP-PKA pathway acts in selective synergism with glucose and tolbutamide to initiate [Ca2+]i signals in pancreatic β-cells. cAMP appears to regulate β-cell sensitivity to glucose and tolbutamide. In contrast, cAMP fails to cooperate with high KCl to increase [Ca2+]i. It is suggested that cAMP acts mainly on a site that is more proximal but functionally linked to the L-type Ca2+ channel, thereby finally increasing Ca2+ influx through this channel.

Control of catecholamine release and blood pressure with octreotide in a patient with pheochromocytoma: a case report with in vitro studies.

A 65-year-old male patient with pheochromocytoma, whose hypertensive episodes were uncontrolled using conventional therapy, was successfully treated with octreotide (SMS 201-995). The serum catecholamine level and the urinary excretion of catecholamines decreased after 300 μg/day of octreotide was administered. To clarify the mechanisms of octreotide that lower catecholamine released from a tumor, we studied the in vitro effects of octreotide on membrane potentials and voltage-dependent Ca2+ channel (VDCC) current using the whole-cell patch-clamp technique in single pheochromocytoma cells dispersed after tumor resection. The action potentials were reversibly inhibited with 10 μM octreotide. In addition, the VDCC current evoked by depolarized pulses from the holding potential of –60 mV was inhibited with 10 μM octreotide. Octreotide is useful for controlling blood pressure before surgery in some patients with uncontrolled hypertension caused by a pheochromocytoma.

Two distinct modes of Ca2+ signalling by ACh in rat pancreatic beta‐cells: concentration, glucose dependence and Ca2+ origin.

1. Calcium signalling by acetylcholine (ACh) in single rat pancreatic beta‐cells was studied. The cytosolic free Ca2+ concentration ([Ca2+]i) was measured by dual‐wavelength fura‐2 microfluorometry. 2. In the presence of basal glucose (2.8 mM), 10(‐6) to 10(‐4) M ACh (high ACh) transiently increased [Ca2+]i. The [Ca2+]i response to 10(‐5) M ACh was little altered under Ca(2+)‐free conditions. Brief pulses of 10(‐5) M ACh evoked successive [Ca2+]i responses, which were progressively inhibited by 0.2‐0.5 microM thapsigargin, a specific inhibitor of the endoplasmic reticulum (ER) Ca2+ pump. 3. Elevation of glucose to 8.3 mM, a concentration which stimulates insulin release, increased [Ca2+]i to an initial peak followed by a sustained, moderate elevation. Addition of 10(‐8) to 10(‐7) M ACh (low ACh) evoked a further increase in [Ca2+]i. The [Ca2+]i response to 10(‐7) M ACh was completely inhibited under Ca(2+)‐free conditions by 1 microM nitrendipine, a blocker of L‐type Ca2+ channels, and by 100 microM diazoxide, an opener of ATP‐sensitive K+ channels. 4. In the presence of 8.3 mM glucose, [Ca2+]i responses to 10(‐5) M ACh were reduced but not abolished by Ca(2+)‐free conditions, nitrendipine and diazoxide. Successive [Ca2+]i transients induced by 10(‐5) M ACh pulses in the presence of nitrendipine were progressively inhibited by thapsigargin. 5. The results revealed two distinct modes of Ca2+ signalling: low ACh increases [Ca2+]i by stimulating Ca2+ influx through voltage‐dependent L‐type Ca2+ channels only in the beta‐cells in which glucose has already elevated [Ca2+]i, while high ACh increases [Ca2+]i at basal as well as stimulatory glucose concentrations by releasing Ca2+ from the ER. The former mechanism is likely to relate to the potentiator action and the latter to the initiator action of ACh on insulin release. High ACh and elevated glucose provoke both modes of Ca2+ signalling.

Current Status of PACAP as a Regulator of Insulin Secretion in Pancreatic Isletsa

PACAP-27 and PACAP-38 as low as 10(-13) M stimulate insulin release from rat islets in a glucose-dependent manner. PACAP also glucose dependently increases cAMP and [Ca2+]i in rat islet beta cells. The [Ca2+]i and insulin secretory responses to PACAP exhibit a similar concentration-response relationship, exhibiting a peak at 10(-13) M. When the [Ca2+]i response is abolished by nitrendipine, a blocker of L-type Ca2+ channels, the insulin response is also inhibited. Insulinotropic peptides glucagon, GLP-1, and VIP also increase [Ca2+]i in beta cells, but only in the nanomolar concentration range. PACAP is 4 logs more potent that VIP, a peptide that exhibits 68% amino acid homology and shares the type II PACAP receptor with PACAP. Immunoreactivity for the type I PACAP receptor is demonstrated in rat islets. Furthermore, PACAP immunoreactivity is demonstrated in nerve fibers and islets in rat pancreas. Based on these findings, we can draw the following conclusions: (1) PACAP is localized in pancreatic nerve fibers and islets; (2) PACAP in the subpicomolar range stimulates insulin release from islets; (3) the stimulation of insulin release is mediated by the cAMP-dependent increase in [Ca2+]i in beta cells; (4) all the PACAP effects are glucose-dependent; (5) PACAP is the most potent insulinotropic hormone known, and (6) the type I PACAP receptor appears to mediate the action of PACAP in the subpicomolar range. Finally, we hypothesize that PACAP is a pancreatic peptide of both neural and islet origin and functions as an intrinsic potentiator of glucose-induced insulin secretion in pancreatic islets (FIG 6).

A case of renal juxtaglomerular cell tumor: usefulness of segmental sampling to prove autonomic secretion of the tumor.

A 27-year-old female patient had been treated for hypertension with conventional therapy for years, because renal vein renin levels failed to show lateralization in renal venous samplings and a renal juxtaglomerular cell tumor (RJGCT) had gone undiagnosed. Abdominal computed tomography revealed a mass at the middle of the right kidney. The right renal venogram demonstrated distinct segmental veins from the upper pole and from the middle and lower poles in the right kidney. On segmental renin sampling from each renal vein, the plasma renin concentration (PRC) of the segmental veins from the middle and lower poles was higher than that from other sites. We diagnosed RJGCT of the right kidney and performed right-sided nephrectomy. After the resection, the PRC rapidly decreased. Immunohistochemical studies using antihuman renin antibodies revealed positive staining of the tumor cells. It is an important strategy to make a segmental sampling at the site as close as possible to the RJGCT.

PACAP as low as 10(-13) M raises cytosolic Ca2+ activity in pancreatic B-cells by augmenting Ca2+ influx through L-type Ca2+ channels to trigger insulin release.

Pancreatic insulin secretion is under control by peptides, as well as by nutrients and other substances(1,2). Truncated glucagon like peptide-1 (tGLP-1), gastric inhibitory peptide (GIP) and glucagon, the members of glucagon/VIP/secretin family of peptides, stimulate insulin release in a low concentration range around 10−9 M, and are thought to be involved in the physiological regulation of insulin release(3–5).

Glucose-insensitivity induced by Ca2+ toxicity in islet β-cells and its prevention by PACAP

Abstract The present study examined whether a sustained increase in cytosolic Ca 2+ concentration ([Ca 2+ ] i ) causes glucose-insensitivity in β-cells and whether it could be modulated by pituitary adenylate cyclase-activating polypeptide (PACAP), a pancreatic insulinotropin. Rat single β-cells were cultured for 2 days with sustained increases in [Ca 2+ ] i , followed by determination of the [Ca 2+ ] i response to glucose (8.3 mM) as monitored with fura-2. High K + (25 mM) produced sustained increases in [Ca 2+ ] i in β-cells, which were inhibited by nifedipine, a Ca 2+ channel blocker. After culture with high K + , the incidence and amplitude of [Ca 2+ ] i responses to glucose were markedly reduced. This glucose-insensitivity was prevented by the presence of nifedipine or PACAP-38 (10 −13 M and 10 −9 M) in high K + culture. PACAP-38 attenuated high K + -induced [Ca 2+ ] i increases. In conclusion, sustained increases in [Ca 2+ ] i induce glucose-insensitivity (Ca 2+ toxicity in β-cells) and it is prevented by PACAP possibly in part due to its Ca 2+ -reducing capacity.

Effects of thapsigahgin, an intracellular CA2+ pump inhibitor, on insulin release by rat pancreatic B-cell

This is the first report as to the effects of thapsigargin (Tg), an inhibitor of intracellular Ca2+ pumps, on insulin release by pancreatic B-cells. Tg does not alter basal insulin release by the isolated islets, with 3 mM glucose. However, it potentiates high glucose-induced insulin release: potentiation of the first phase response is dose-related in a concentration range of 1.3-40 microM. In isolated B-cells, Tg causes a minimal rise in basal cytosolic free calcium concentration ([Ca2+]i) and eliminates high glucose-induced initial lowering of [Ca2+]i. Tg does not alter glucose oxidation by the islets and the islet insulin content. An elimination of glucose-induced sequestration of Ca2+ into Tg-sensitive intracellular pool(s) is considered to be the cause of Tg potentiation of glucose effect on insulin release.

Cyclic AMP, a Mediator of GLP-1, Acts in Concert with Glucose and Tolbutamide to Increase Cytosolic Ca2+ in Pancreatic B-Cells

Cyclic AMP (cAMP) plays an important role in the regulation of islet B-cell function and stimulates insulin release in a glucose-dependent manner(1). Several physiologic peptides such as truncated glucagon-like peptide-1 (GLP-1), gastric inhibitory peptide (GIP) and glucagon are known to elevate cAMP level in islets and stimulate insulin release also in a glucose-dependent manner(1–4). It has been recently shown that both a rise in cAMP and these peptides increase the cytosolic free Ca2+ concentration ([Ca2+]i) in pancreatic B-cells(3–7), the key intracellular signal that triggers exocytosis of insulin(8,9). The increase in [Ca2+]i is also glucose-dependent(6) and is tightly linked to insulin release(3). However, the mechanisms for the glucose-dependent action of cAMP to increase [Ca2+]i is not well understood. Whether cAMP increases [Ca2+]i via specific cooperation with glucose, a fuel secretagogue, or also with other types of insulin secretagogues is of particular importance. Whether the glucose-dependent action of the peptides to increase [Ca2+]i is mediated by a rise in cAMP is not thoroughly determined.