Effects of relaxin on mast cells. In vitro and in vivo studies in rats and guinea pigs.

The results of the current study demonstrate that relaxin inhibits histamine release by mast cells. This effect is related to the peptide concentrations, and could be observed in both isolated rat serosal mast cells stimulated with compound 48/80 or calcium ionophore A 23187, and in serosal mast cells isolated from sensitized guinea pigs and challenged with the antigen. The morphological findings agree with the functional data, revealing that relaxin attenuates calcium ionophore-induced granule exocytosis by isolated rat serosal mast cells. Similar effects of relaxin have also been recognized in vivo by light microscopic and densitometric analysis of the mesenteric mast cells of rats which received the hormone intraperitoneally 20 min before local treatment of the mesentery with calcium ionophore. Moreover, evidence is provided that relaxin stimulates endogenous production of nitric oxide and attenuates the rise of intracellular Ca2+ concentration induced by calcium ionophore. The experiments with drugs capable of influencing nitric oxide production also provide indirect evidence that the inhibiting effect of relaxin on mast cell histamine release is related to an increased generation of nitric oxide. It is suggested that relaxin may have a physiological role in modulating mast cell function through the L-arginine-nitric oxide pathway.

Relaxin activates the L-arginine-nitric oxide pathway in vascular smooth muscle cells in culture.

The peptide hormone relaxin (RLX) has been shown to elicit a powerful vasodilatory response in several target organs. This response is mediated by the stimulation of intrinsic nitric oxide (NO) generation. The present study was designed to clarify whether RLX directly promotes the relaxation of vascular smooth muscle cells through stimulation of NO generation. Vascular smooth muscle cells from bovine aortas were incubated with RLX at concentrations ranging from 1 nmol/L to 1 micromol/L. The expression and activity of NO synthase, production of NO, and the intracellular levels of cGMP and Ca2+ were determined. The cell morphology and signal transduction mechanisms of these bovine aortic smooth muscle cells in response to RLX were also studied. RLX stimulated the expression of immunoreactive inducible NO synthase and increased significantly and in a concentration-related fashion inducible NO synthase activity, NO generation, and intracellular cGMP levels. Concurrently, RLX significantly decreased cytosolic Ca2+ concentrations and caused changes in cell shape and the actin cytoskeleton that were consistent with cell relaxation. The signal transduction mechanisms leading to the enhanced expression of inducible NO synthase protein and activity caused by RLX involve the activation of tyrosine kinase, phosphatidylcholine-phospholipase C, and the transcription factor nuclear factor-kappaB, similar to bacterial endotoxins and proinflammatory cytokines. This study suggests that RLX is an endogenous agent capable of regulating vascular tone by activation of the L-arginine-NO pathway in vascular smooth muscle cells.

Immunocytochemical and ultrastructural changes of islet cells in rats treated long-term with cyclosporine at immunotherapeutic doses.

Daily cyclosporine doses of 10 mg/kg body weight for 21 days in Wistar rats cause impairment in glucose homeostasis and changes in the amount of immunostainable hormones and in the ultrastructure of the cells of the pancreatic islets. CsA induces hyperglycemia and reduced glucose tolerance, and causes a decrease in immunoreactive insulin and an increase of somatostatin and pancreatic polypeptide (PP) immunoreactivities, leaving glucagon immunoreactivity unaffected. Ultrastructurally, different degrees of dilation of rough endoplasmic reticulum cisternae and enlargement of Golgi apparatus can be observed in B cells, together with a pronounced reduction in the number of secretory granules. Nevertheless, there were no apparent morphological changes of the other cytoplasmic organelles, suggesting that the drug, besides a depression of protein synthesis, as previously stated, also induces a substantial defect in granulogenesis, probably due to impairment in the intracellular transport of the hormone from the sites of synthesis to the secretory granules. The B cell alterations are not accompanied by any sign of B cell degeneration or death. Non-B cells did not show any of the ultrastructural changes found in B cells and were similar to those of the control rats. The above findings indicate that CsA at immunotherapeutic doses causes impairment in the secretory processes of B cells specifically. An hypothesis on the mode of action of CsA on B cells is drawn.

Relaxin-induced increased coronary flow through stimulation of nitric oxide production.

1. Relaxin (RLX) is a multifunctional hormone which, besides its role in pregnancy and parturition, has also been shown to influence the cardiovascular system. In this study, we investigated the effect of RLX on coronary flow of rat and guinea-pig hearts, isolated and perfused in a Langendorff apparatus. RLX was either added to the perfusion fluid at a concentration of 5 x 10(-9) M for a 20-min perfusion, or given as a bolus into the aortic cannula at concentrations of 10(-9) M, 5 x 10(-8) M dissolved in 1 ml of perfusion fluid. 2. RLX, given either for a 20-min perfusion or as a bolus in the aortic cannula to guinea-pig and rat isolated hearts, increased the coronary flow and the amount of nitrite, a stable end-product of nitric oxide (NO) metabolism, that appeared in the perfusates in a concentration-dependent fashion. 3. The increase in coronary flow and in nitrite in the perfusates induced by RLX was significantly reduced by pretreatment with the nitric oxide synthase (NOS) inhibitor, NG-monomethyl-L-arginine (L-NMMA, 10(-4) M). 4. The effects of RLX on coronary flow and nitrite amounts in the perfusates were compared with those induced by the endothelium-dependent vasodilator agent, acetylcholine (ACh, 10(-8)-10(-7) M), and by the endothelium-independent vasodilator agent, sodium nitroprusside (SNP, 10(-7)-10(-6) M). The results obtained show that RLX is more effective than ACh and SNP in increasing coronary flow. 5 The results of this study show that RLX increases coronary flow through stimulation of NO production; hence this hormone should be regarded as a novel agent capable of improving myocardial perfusion.

The vasorelaxant hormone relaxin induces changes in liver sinusoid microcirculation: a morphologic study in the rat.

This study shows that specialized contractile endothelial cells exist in rat liver sinusoids which may be involved in the local control of hemodynamics and which are sensitive to vasoactive agents, including the vasorelaxant hormone relaxin. Male rats were treated with 10 microg relaxin for 4 days; phosphate-buffered saline (PBS)-treated rats were the controls. For comparison, rats treated with relaxin together with the NO-synthase inhibitor N(omega)-nitro-l -arginine methyl ester (L-NAME), and rats treated with the vasodilator taurodeoxycholic acid or the vasoconstrictor ethanol were investigated. Liver fragments were studied morphologically and morphometrically. In the control rats, peculiar contractile cells were present in the endothelial lining. These cells had abundant myofilaments and formed cytoplasmic blebs projecting into and often occluding the lumen. In the ethanol-treated rats, sinusoids were constricted and filled with cytoplasmic blebs. In the relaxin-treated rats, sinusoids were markedly dilated and the cytoplasmic blebs nearly disappeared. Similar findings were observed in the taurodeoxycholic acid-treated rats. The effects of relaxin were blunted by L-NAME, suggesting that the relaxin action involves an NO-mediated mechanism.

A multihormonal tumor of the pancreas producing neurotensin associated with the WDHA syndrome. Histology, histochemistry and origin.

A pancreatic tumor associated with severe WDHA syndrome has been studied histologically and immunohistochemically. Light microscopy revealed that the growth pattern of the tumor varied greatly from zone to zone but with prevailing solid arrangement of the tumoral cells. The majority of the endocrine cells showed numerous eosinophilic, PTAH-positive, and argyrophilic secretory granules, that were ultrastructurally similar to those of normal and tumoral neurotensin-containing cells. A minority of the endocrine cells had secretory granules ultrastructurally different from the aforementioned ones, but these were not diagnostic on purely morphological grounds. Inside the tumor, immunohistochemistry demonstrated a majority of neurotensin-immunoreactive cells, sparse and small clusters of VIP-immunoreactive cells and few, dispersed pancreatic polypeptide-immunoreactive cells. Some structural and ultrastructural aspects of the tumoral stroma have also been reported. Ducts and solid masses of duct-like cells were also found, and small clusters and singly dispersed duct-like cells were seen invading the endocrine tissue and undergoing mitoses. Such features suggest that the tumor originated from precursors located in the medium-sized and small pancreatic ducts. Because of the multihormonal nature of the tumor, the role of neurotensin and VIP in producing the patient's symptoms is discussed and a synergistic action of the two hormones is suggested in causing the particularly severe WDHA syndrome.

Effects of relaxin on the microvasculature of mouse mammary gland.

The effects of RLX on the microvasculature of the mouse mammary gland are reported. RLX (pure porcine standard NIH-RXN-P1) at a dose of 3 GPU was administered subcutaneously to virgin adult mice ovariectomized 12 days before. The mammary glands were removed 18-20 h after RLX injection and their examination by light microscopy did not reveal any substantial growth-response to the hormone. Histology and morphometry indicated striking dilation of microvessels, especially capillaries, and electron microscopy revealed an increase in the micropinocytotic vesicles, thus suggesting enhanced transendothelial transport of substances. Such phenomena, which were independent of a release of granules by mast cells, may represent an important component of the mammotrophic action of RLX.

Relaxin promotes differentiation of human breast cancer cells MCF-7 transplanted into nude mice.

Previous studies showed that the hormone relaxin acts on human breast cancer MCF-7 cells in vitro by modulating cell proliferation and promoting cell differentiation toward a duct epithelial phenotype. The present study was designed to investigate whether relaxin retains these properties when acting in vivo on MCF-7 cell tumors developed in athymic nude mice. Mice bearing MCF-7 cell tumors transplanted under the mammary fat pad and estrogenized to sustain tumor growth were treated systemically with relaxin (10 microg/day) for 19 days. Vehicle-treated mice were used as controls. Thirty days later, the mice were sacrificed and tumor fragments were analyzed by light and electron microscopy and immunocytochemistry. Measurements of tumor volume were recorded weekly for the overall experimental period. The results obtained indicate that relaxin treatment promotes differentiation of tumor cells towards both myoepithelial-like and epithelial-like cells, as judged by the ultrastructural features of the cells and by the increased expression of smooth muscle actin and cadherins. Measurements of tumor size and of the number of cycling cells show that relaxin, at the doses and times of exposure used in this study, does not significantly influence tumor growth and cell proliferation.

Are pancreatic VIPomas paraneuron neoplasms? A clue to the neuroectodermal origin of these tumors.

Three pancreatic vasoactive intestinal polypeptide (VIP)-producing tumors associated with the watery diarrhea-hypokalemia-achlorhydria syndrome were studied histologically, ultrastructurally, and immunocytochemically. All the tumors contained varying numbers of cells arranged in pseudoglandular structures. The cells showed a polar organization, with apical tuft of microvilli and basal VIP-containing, synaptic vesicle-like granules. Based on the morphology of the VIPoma cells typical of recepto-secretory cells, together with the ability to synthesize and release a peptide that in normal conditions is expressed exclusively by neurons, and the absence of VIP-producing endocrine cells in normal pancreas and gastrointestinal mucosa, the hypothesis is drawn that the pancreatic VIPomas reported here are paraneuron neoplasms, which possibly originate from neuroectodermal ancestors.

Immunocytochemical and ultrastructural abnormalities of islet tissue in patients with VIP-producing tumors of the pancreas.

Nontumoral endocrine pancreas from three patients with malignant vasoactive intestinal polypeptide (VIP)-omas and the Verner-Morrison (watery diarrhea, hypokalemia, and hypoachlorhydria) syndrome was studied immunocytochemically, ultrastructurally, and morphometrically. Compared with normal islets from control subjects, those of the VIPoma-associated pancreas showed a decrease of immunoreactive insulin in B-cells associated with cytological features indicative of enhanced insulin synthesis and secretion and an increase in the number of immunoreactive somatostatin- and pancreatic polypeptide-containing cells, in the absence of ultrastructural signs of modified secretory activity. No substantial alterations of A-cells were observed. In addition, images of diffuse de novo formation of ducts and islet tissue were often found. Possible mechanisms involved in determining the above changes are discussed.

A malignant tumor of the pancreas producing glucagonoma syndrome: immunocytochemistry and ultrastructure of liver metastases and comparison with the primary tumor.

In this study, liver metastases from a patient with a pancreatic glucagonoma producing the syndrome have been investigated histologically, ultrastructurally, and immunocytochemically. A comparison has also been made between the metastases and the primary pancreatic tumor investigated in a parallel study. In the metastatic tissue, glucagon-, pancreatic polypeptide (PP)-, and somatostatin-containing cells were found together with a majority of cells without any immunoreactivity. Glucagon-positive cells were much more numerous than PP- and somatostatin-immunoreactive cells. As in the primary tumor, double immunogold staining of ultrathin sections demonstrated the co-existence of glucagon and PP immunoreactivities in most of the granulated cells, but PP immunolabeling was often faint, so that it probably could not be revealed by the PAP method in light microscopical sections. Such a finding, together with the histological and ultrastructural features, is consistent with an ontogenic and phylogenetic primitiveness of the metastatic cell population.

A malignant tumor of the pancreas producing glucagonoma syndrome: coexistence of glucagon and pancreatic polypeptide (PP) in the tumor cells.

A malignant tumor of the pancreas producing the glucagonoma syndrome and associated with high plasma levels of glucagon and pancreatic polypeptide was studied histologically, ultrastructurally, and immunocytochemically. The histologic and ultrastructural features were closely similar to those of previously reported malignant glucagonomas. However, immunolabeling with specific antisera revealed that, in addition to cells having only glucagon- or only pancreatic polypeptide-immunoreactivity, other cells were also present, showing a co-existence of both peptides. These findings indicate that the tumor contains a cell population with a phenotype similar to that of intestinal L-cells rather than to pancreatic A-cells.

Presence of functionally active beta-adrenoceptors in rat mast cells. Correlation between (--)[3H]-dihydroalprenolol binding and inhibition of histamine release.

The correlation between the binding of a beta-adrenoceptor antagonist, (--)[3H]-dihydroalprenolol (DHAP), and the adrenergic inhibition of histamine release by acetylcholine and by compound 48/80 was studied with isolated purified rat mast cells and in rat mast cell crude membrane fractions. Acetylcholine-evoked histamine release was inhibited by catecholamines, in the order isoprenaline greater than adrenaline greater than noradrenaline. Pretreatment of cells with (--)alprenolol antagonized the inhibitory effect of isoprenaline on acetylcholine-induced histamine release. 40/80-evoked histamine release was blocked by isoprenaline at significantly higher concentrations than those required to inhibit cholinergic histamine release. The inhibitory effect of isoprenaline was equally antagonized by preincubating mast cells with (--)alprenolol. Specific binding sites for DHAP have been demonstrated in rat mast cell membranes. The specific binding of DHAP was inhibited by adrenoceptor agonists and antagonists according to the stereospecificity of these compounds. A close correlation between the binding-inhibitory potency of various adrenergic compounds and the data obtained in the pharmacological experiments was found, thus indicating the presence of beta-adrenoceptors in rat mast cells.