Olof Larsson

Co-existence of peptide HI (PHI) and VIP in nerves regulating blood flow and bronchial smooth muscle tone in various mammals including man

By immunohistochemistry it was found that PHI- and VIP-like immunoreactivity (-IR) occurred in the same autonomic neurons in the upper respiratory tract, tongue and salivary glands with associated ganglia in rat, guinea-pig, cat, pig and man. VIP- and PHI-like immunoreactivity was also found in similar locations in the human heart. The N-terminally directed, but not the C-terminally directed, PHI antiserum or the VIP antiserum stained endocrine cells in the pig duodenum. This suggests the existence of an additional PHI-like peptide. Ligation of nerves acutely caused marked overlapping axonal accumulations of PHI- and VIP-IR central to the lesion. Two weeks after transection of the nerves, both types of immunoreactivities were still observed in accumulations both in the axons as well as in the corresponding cell bodies. The levels of PHI- and VIP-IR in normal tissues from the cat were around 10-50 pmol/g with a molar ratio of about 1 to 2. Systemic administrations of PHI and VIP induced hypotension, probably due to peripheral vasodilation in both guinea-pig and cat. Furthermore, both PHI and VIP caused an inhibition of the vagally induced increase in respiratory insufflation pressure in guinea-pig. PHI and VIP relaxed the guinea-pig trachea in vitro, suggesting a direct action on tracheobronchial smooth muscle. VIP was about 5-10 times more potent than PHI with regard to hypotensive effects and 2-3-fold, considering respiratory smooth muscle-relaxant effects in the guinea-pig. PHI was about 50-fold less potent to induce hypotension in the cat than in the guinea-pig. Although species differences seem to exist as regards biological potency, PHI should also be considered when examining the role of VIP as an autonomic neurotransmitter.

PKC-dependent stimulation of exocytosis by sulfonylureas in pancreatic beta cells.

Hypoglycemic sulfonylureas represent a group of clinically useful antidiabetic compounds that stimulate insulin secretion from pancreatic β cells. The molecular mechanisms involved are not fully understood but are believed to involve inhibition of potassium channels sensitive to adenosine triphosphate (KATP channels) in the β cell membrane, causing membrane depolarization, calcium influx, and activation of the secretory machinery. In addition to these effects, sulfonylureas also promoted exocytosis by direct interaction with the secretory machinery not involving closure of the plasma membrane KATP channels. This effect was dependent on protein kinase C (PKC) and was observed at therapeutic concentrations of sulfonylureas, which suggests that it contributes to their hypoglycemic action in diabetics.

Neuropeptide Y receptor in pig spleen: binding characteristics, reduction of cyclic AMP formation and calcium antagonist inhibition of vasoconstriction.

Specific, high-affinity binding sites for 125I-porcine neuropeptide Y (NPY) were demonstrated in membranes from the pig spleen. The equilibrium dissociation constant (KD) of the receptor 125I-NPY complex was 532 +/- 87 pM and the maximal number of specific binding sites (Bmax) 23 +/- 3 fmol/mg protein. The Scatchard plot for 125I-NPY binding under equilibrium conditions showed a best-fit to a straight line, whereas the dissociation appeared biphasic. 125I-NPY binding was unaffected by adrenoceptor antagonists and was inhibited by the guanosine triphosphate (GTP) analogue guanylylimidodiphosphate, suggesting regulation by a GTP binding protein. A series of NPY analogues showed a good correlation between binding, inhibition of forskolin-induced cyclic adenosine monophosphate (cAMP) formation and vasoconstrictor activity in vivo. A large carboxyl terminal portion of NPY and the carboxyl terminal amide were essential for binding, inhibition of cAMP formation and vasoconstrictor effects. The NPY fragment 13-36, which has been reported to act only on prejunctional NPY receptors, showed only a 10-fold lower potency than NPY-(1-36) both in binding to splenic membranes and vasoconstrictor activity in vivo. Phenylephrine increased phosphatidyl inositol turnover whereas NPY-(1-36) or -(13-36) did not induce formation of inositol phosphates. The calcium antagonists felodipine and nifedipine attenuated the splenic vasoconstrictor response to NPY in vivo but not the NPY-evoked inhibition of cAMP accumulation or the specific binding of 125I-NPY.(ABSTRACT TRUNCATED AT 250 WORDS)

Imidazoline Compounds Stimulate Insulin Release by Inhibition of KATP Channels and Interaction With the Exocytotic Machinery

A novel imidazoline compound, RX871024, was used to investigate the mechanisms by which imidazoline derivatives promote insulin secretion in rat pancreatic β-cells and HIT T15 cells. RX871024 stimulated insulin release from rat pancreatic β-cells and HIT T15 cells in a glucose-dependent way. This effect was not related to α2-adrenergic, I1-, and I2-imidazoline receptors. RX871024 promoted insulin release by at least two modes of action. One included an increase in cytoplasmic free Ca2+ concentration ([Ca2+]i), subsequent to blocking of ATP-dependent K+ channels, membrane depolarization, and activation of voltage-dependent Ca2+ channels. The other, a more distal effect of imidazoline, affected the exocytotic machinery and was unrelated to changes in membrane potential and [Ca2+]i. The mechanism of RX871024-induced insulin release was dependent on protein kinases A and C. The sensitizing effect of a low dose of RX871024 on glucose-induced insulin secretion suggests that imidazoline compounds of this kind may constitute the basis for development of a new class of oral hypoglycemic agents.

Cysteine string protein (CSP) is an insulin secretory granule-associated protein regulating beta-cell exocytosis.

Cysteine string proteins (CSPs) are novel synaptic vesicle‐associated protein components characterized by an N‐terminal J‐domain and a central palmitoylated string of cysteine residues. The cellular localization and functional role of CSP was studied in pancreatic endocrine cells. In situ hybridization and RT–PCR analysis demonstrated CSP mRNA expression in insulin‐producing cells. CSP1 mRNA was present in pancreatic islets; both CSP1 and CSP2 mRNAs were seen in insulin‐secreting cell lines. Punctate CSP‐like immunoreactivity (CSP‐LI) was demonstrated in most islets of Langerhans cells, acinar cells and nerve fibers of the rat pancreas. Ultrastructural analysis showed CSP‐LI in close association with membranes of secretory granules of cells in the endo‐ and exocrine pancreas. Subcellular fractionation of insulinoma cells showed CSP1 (34/36 kDa) in granular fractions; the membrane and cytosol fractions contained predominantly CSP2 (27 kDa). The fractions also contained proteins of 72 and 70 kDa, presumably CSP dimers. CSP1 overexpression in INS‐1 cells or intracellular administration of CSP antibodies into mouse ob/ob β‐cells did not affect voltage‐dependent Ca2+‐channel activity. Amperometric measurements showed a significant decrease in insulin exocytosis in individual INS‐1 cells after CSP1 overexpression. We conclude that CSP is associated with insulin secretory granules and that CSP participates in the molecular regulation of insulin exocytosis by mechanisms not involving changes in the activity of voltage‐gated Ca2+‐channels.

Corelease of vasoactive intestinal polypeptide and peptide histidine isoleucine in relation to atropine-resistant vasodilation in cat submandibular salivary gland

Parasympathetic nerve stimulation of the submandibular salivary gland in the cat caused salivary secretion, vasodilation and a corelease of vasoactive intestinal polypeptide (VIP) and peptide histidine isoleucine (PHI) immunoreactivities (IR) into the venous effluent, as indicated by an increase in output. The ratio between the released VIP-IR and PHI-IR was close to 1:1. Gel-permeation chromatography of plasma from the submandibular venous effluent indicated that the released VIP-IR and PHI-IR were very similar to porcine VIP and PHI, respectively. Atropine pretreatment enhanced output of both VIP-IR and PHI-IR during the parasympathetic nerve stimulation to a similar extent (about 5-fold) compared to control stimulations. This increase could be due to an inhibitory presynaptic muscarinic receptor regulation of VIP and PHI release. Since VIP and PHI are present in the same postganglionic parasympathetic nerves in the gland and both peptides have vasodilator activity, the present data suggest that both VIP and PHI may contribute to the atropine-resistant vasodilation seen upon stimulation of the chorda-lingual nerve. The parasympathetic control of salivary gland function may thus involve, a multimessenger system with the classical transmitter acetylcholine and the peptides VIP and PHI.

Distinct pharmacologic properties of neuromuscular blocking agents on human neuronal nicotinic acetylcholine receptors: a possible explanation for the train-of-four fade.

Background:Nondepolarizing neuromuscular blocking agents (NMBAs) are extensively used in the practice of anesthesia and intensive care medicine. Their primary site of action is at the postsynaptic nicotinic acetylcholine receptor (nAChR) in the neuromuscular junction, but their action on neuronal nAChRs have not been fully evaluated. Furthermore, observed adverse effects of nondepolarizing NMBAs might originate from an interaction with neuronal nAChRs. The aim of this study was to examine the effect of clinically used nondepolarizing NMBAs on muscle and neuronal nAChR subtypes. Methods:Xenopus laevis oocytes were injected with messenger RNA encoding for the subunits included in the human &agr;1&bgr;1ϵ&dgr;, &agr;3&bgr;2, &agr;3&bgr;4, &agr;4&bgr;2, and &agr;7 nAChR subtypes. The interactions between each of these nAChR subtypes and atracurium, cisatracurium, d-tubocurarine, mivacurium, pancuronium, rocuronium, and vecuronium were studied using an eight-channel two-electrode voltage clamp setup. Responses were measured as peak current and net charge. Results:All nondepolarizing NMBAs inhibited both muscle and neuronal nAChRs. The neuronal nAChRs were reversibly and concentration-dependently inhibited in the low micromolar range. The mechanism (i.e., competitive vs. noncompetitive) of the block at the neuronal nAChRs was dependent both on subtype and the NMBA tested. The authors did not observe activation of the nAChR subtypes by any of the NMBAs tested. Conclusions:The authors conclude that nondepolarizing NMBAs concentration-dependently inhibit human neuronal nAChRs. The inhibition of the presynaptic &agr;3&bgr;2 nAChR subtype expressed at the motor nerve ending provides a possible molecular explanation for the tetanic and train-of-four fade seen during a nondepolarizing neuromuscular block.

Effects of VIP, PHM and substance P on blood vessels and secretory elements of the human submandibular gland

The effects of the neuropeptides VIP, PHM and substance P (SP) on vascular smooth muscle tone, K+ secretion from exocrine elements and tissue content of cyclic AMP (cAMP) in the human submandibular gland were studied in vitro. All three peptides caused relaxation of noradrenaline contracted human submandibular arteries at nM concentrations. SP was slightly more active than VIP and PHM which had a similar potency as vasodilators. Only carbachol but not VIP, PHM or SP stimulated K+ secretion from exocrine elements of the human submandibular gland. Principally similar in vitro effects on K+ secretion were obtained on the cat submandibular gland, but in the rat not only carbachol but also SP stimulated K+ secretion. VIP and PHM increased cAMP production of exocrine elements in the human submandibular gland in nM concentrations. VIP was about 5-fold more potent than PHM with regards to cAMP production. In conclusion, VIP, PHM and SP relaxed human submandibular arteries in vitro. Both VIP and PHM stimulated cAMP production in glandular tissue but none of the three peptides induced K+ secretion from human submandibular gland tissue. This suggests that, in contrast to the situation in the rat, SP does not cause watery salivation in man, while VIP and PHM may modulate protein e.g. amylase content of the saliva.

Impaired Coupling of Glucose Signal to the Exocytotic Machinery in Diabetic GK Rats

The GK rat is a spontaneous model of NIDDM. The insulin response to 16.7 mmol/l glucose was markedly impaired in both isolated perfused pancreas and isolated islets from GK rats compared with control Wistar rats. Depolarization with 30 mmol/l KC1 in the presence of 3.3 mmol/l glucose and 250 μmol/l diazoxide induced similar insulin responses in perfused pancreases of GK and control rats. In contrast, the glucose-stimulated insulin release was also severely impaired in GK pancreases in the depolarized state. Forskolin (1 μmol/l) markedly enhanced insulin release at 3.3 mmol/l glucose in GK but not control pancreases (54 ± 15 vs. 3 ± 1 pmol/l0 min, P < 0.001). Dibutyryl cAMP (1 mmol/l) exerted effects similar to forskolin on insulin release in the perfused pancreas. In depolarized pancreases of GK but not control rats, forskolin also induced a marked insulin response at 3.3 mmol/l glucose (163 ± 48 vs. 16 ± 1 pmol/20 min,< P < 0.03). Similarly, in studies on isolated islets from GK rats cultured in 5.5 or 16.7 mmol/l glucose for 48 h, forskolin (5 μmol/l) restored insulin release in response to 16.7 mmol/l glucose but had no effect on islet glucose utilization at 3.3 or 16.7 mmol/l glucose. Forskolin markedly stimulated insulin release at 3.3 mmol/l glucose in GK but not control rat islets cultured for 48 h in 5.5 mmol/l glucose, whereas 20 mmol/l arginine had an almost identical effect in both islet varieties. However, in islets cultured in 16.7 mmol/l glucose, forskolin stimulated insulin release similarly both in control and GK islets at 3.3 mmol/l glucose. In conclusion, this study suggests that the insulinotropic effects of glucose are coupled to a direct regulation of the exocytotic machinery in the pancreatic 3-cell. This pathway is markedly impaired in GK rats, contributing to defective insulin response to glucose. In this model, cAMP generation restores the insulin response to 16.7 mmol/l glucose and exerts a marked insulin release even at 3.3 mmol/l glucose.

Separate processes mediate nucleotide-induced inhibition and stimulation of the ATP-regulated K(+)-channels in mouse pancreatic beta-cells.

The mechanisms by which nucleotides stimulate the activity of the ATP regulated K+-channel (KATP-channel) were investigated using inside-out patches from mouse pancreatic β-cells. ATP produces a concentration-dependent inhibition of channel activity with a K1 of 18 μM. The inhibitory action of ATP was counteracted by ADP (0.1 mM) and GDP (0.2 mM) but not GTP (1 mM). Stimulation of channel activity was also observed when ADP, GDP and GTP were applied in the absence of ATP. The ability of ADP and GDP to reactivate KATP-channels blocked by ATP declined with time following patch excision and after 30-60 min these nucleotides were without effect. During the same time period the ability of ADP and GTP to stimulate the channel in the absence of ATP was lost. In fact, ADP now blocked channel activity with 50% inhibition being observed at approximately 0.1 mM. By contrast, GDP remained a stimulator in the absence of ATP even when its ability to evoke channel activity in the presence of ATP was lost. These observations show that nucleotide-induced activation of the KATP-channel does not involve competition with ATP for a common inhibitory site but involves other processes. The data are consistent with the idea that nucleotides modulate KATP-channel activity by a number of different mechanisms that may include both regulation of cytosolic constituents and direct interaction with the channel and associated control proteins.