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Vascular endothelial growth factor (VEGF), the strongest known angiogenic cytokine and also a potent enhancer of vascular permeability, is closely associated with diabetic ocular complications and other intraocular neovascular diseases. The therapeutic effect of VEGF-neutralizing antibody on oxygen-induced retinopathy in an experimental murine model of proliferative retinopathy was investigated. Intraocular and systemic injection of the antibody resulted in 46% and 18% reductions in the number of nuclei of newly formed vessels of this model, respectively. The results demonstrated that a neutralizing antibody against VEGF was highly effective in the treatment of intraocular neovascularization and suggested possible modes of therapy in human intraocular neovascular diseases, including diabetic proliferative retinopathy.

Vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) is a secreted mitogen for vascular endothelial cells, and it promotes vascular permeability and neovascularization in vivo. We investigated the mechanisms by which low oxygen tension modulates the expression of VEGF in human aortic vascular smooth muscle cells (h-SMC) in vitro. Moreover, we measured VEGF levels in the cultured medium with or without endothelin-1 (ET-1) using a newly developed, highly sensitive, enzyme-linked immunosorbent assay. Hypoxia resulted in a substantial induction of VEGF transcripts at 3 and 24 hr. VEGF levels were significantly higher when h-SMC were cultured in medium containing ET-1 than when cultured in medium without ET-1. In conclusion, hypoxia and ET-1 constitute potent stimuli for VEGF production in h-SMC.

Long-term treatment with n-3 eicosapentaenoic acid (EPA) has been shown to exert hypotensive effects and have beneficial effects on atherosclerosis. To elucidate one of the underlying mechanisms of these effects, intracellular calcium concentration [Ca2+]i, and resting membrane potential were measured in rat vascular smooth muscle cells (A7r5 cell) treated with EPA, using Ca2+-sensitive dye fura-2 AM and the patch clamp technique. The alterations in fatty acid compositions of phospholipids and cell migration after treatment with EPA (30 microM) for 6 h-7 days were also examined. After treating cells with EPA, the EPA and DPA (docosapentaenoic acid) content of the phospholipid fraction (mol.%) increased in a time-dependent manner. Alternatively, arachidonic acid (AA) decreased, and then the ratio of EPA and AA (EPA/AA) increased significantly. The resting [Ca2+]i decreased from 170 +/- 46 nM (n = 16) in control cells to 123 +/- 29 nM (n = 16) in cells treated with EPA (30 microM) for 7 days. Vasopressin (100 nM), endothelin-1 (100 nM) and platelet-derived growth factor (PDGF 5 ng/ml) evoked an initial peak of [Ca2+]i, followed by a smaller sustained rise of [Ca2+]i in the presence of extracellular Ca2+. In EPA-treated cells, both the peak and the sustained rise of [Ca2+]i induced by these agonists decreased in comparison to the control cells. EPA treatment also decreased the transient [Ca2+]i rise evoked by these agonists in the absence of extracellular Ca2+. Under the current clamp condition, resting membrane potential was significantly higher in EPA-treated cells (-49.8 +/- 10.4 mV, n = 41) than in control cells (-44.6 +/- 7.4 mV, n = 41, P < 0.05), and the input resistance of the cell was lower in EPA-treated cells, while cell size and capacitance were not statistically different. In addition, long-term treatment with EPA for 7 days significantly inhibited PDGF-induced cell migration. These results suggest that cellular incorporation of n-3 eicosapentaenoic acid attenuates intracellular mechanisms related to changes of [Ca2+]i and affects membrane potential, thereby inhibiting migration of vascular smooth muscle cells. These actions of EPA may contribute to its vasorelaxant and antiatherosclerotic effects.

Arterial smooth muscle cell migration from the media to the intima is a crucial process in the pathogenesis of atherosclerosis. Platelet-derived growth factor (PDGF) has been proposed to play a key role in the development of advanced atherosclerotic lesions by stimulating the migration and proliferation of vascular smooth muscle cells. Polyunsaturated fatty acids (PUFA) of the omega-3 series, extracted from fish oil has been shown to have beneficial effects on atherosclerosis. In this study, we evaluated the effects of omega-3 PUFA on the migration of human aortic smooth muscle cell (hASMC) in vitro. The migration assay was performed according to the Capsoni's method using transwell culture plates. PDGF, fibrinogen or 10% FCS significantly stimulated hASMC migration, however, omega-3 PUFA significantly inhibited PDGF-induced migration of hASMC. These results suggest that the inhibitory effect of omega-3 PUFA on cell migration may be an important aspect by which omega-3 PUFA exerts its antiatherosclerotic influence.

Messenger RNA of receptor for advanced glycation end products (RAGE) is abundantly expressed in the lung. However, cell types expressing RAGE mRNA in the lung have not been identified. In order to elucidate the function of RAGE in pulmonary tissue, we have identified a cell type expressing RAGE mRNA by in situ hybridization and compared its expression level of RAGE mRNA by RNA blot analysis of isolated cells. In situ hybridization revealed that RAGE mRNA was intensely and specifically visualized in alveolar epithelial type II (AT-II) cells, and weakly in alveolar macrophages. The expression of RAGE mRNA in the primary culture of AT-II cells was at a high level, but that in alveolar macrophages isolated from alveolar lavage was under the level of detection by RNA blot analysis. These results showed that RAGE mRNA is specifically expressed in AT-II cells, and suggested that RAGE makes a substantial contribution to the function of AT-II cells in the lung.

The effects of elevated glucose and aldose reductase inhibitor (ARI:ONO-2235) on nitric oxide (NO) production in cultured human umbilical endothelial cells (HUVEC) were evaluated. Aldose reductase and nitric oxide synthase(NOS) share NADPH as an obligate cofactor, therefore it is suggested that the enhanced of glucose flux (27.5 mM) by aldose reductase inhibited NO production by blunting NOS activity. However, the addition of ONO-2235 (100 μM) prevented the inhibition of [NO 2 − ] production. Since ARI decreases glucose-mediated inhibition of NO production in HUVEC, this agent might ameliorate endothelial function associated with diabetes.

1,5-Anhydroglucitol (1.5-AG) is known to closely reflect diabetic control within several days. The possibility of predicting long-term glycemic control after an educational hospitalization of type II diabetic patients was investigated by examining the relationship between changes in serum 1,5-AG levels after a short-term trial home stay following an educational program and long-term changes in glycosylated hemoglobin A1c (HbA1c) levels after discharge. After 22 patients with type II diabetes had successfully completed the educational hospitalization program, they returned as outpatients for 5 nights in a row. Changes in serum 1,5-AG levels were determined during this period. The HbA1c levels were then determined over a period of 3 months after discharge, and the relationship between changes in 1,5-AG and HbA1c levels was examined. Changes in serum 1,5-AG levels during the 5-day trial home stay and the changes in HbA1c levels during the 3 months after discharge from the hospital were found to be significantly correlated (r = 0.70, P0.01). A comparison of the decreased group, which exhibited a decrease in 1.5-AG levels of 5.0 mumol/l or more during the trial home stay, and the unchanged group, revealed that increases in body mass index 3 months after discharge were significantly higher in the decreased group (1.2 +/- 0.4%) than in the unchanged group (0.2 +/- 0.5%) (P0.05). Determination of serum 1,5-AG levels of patients with type II diabetes before and after a trial home stay following educational hospitalization was found to be useful in identifying patients at high risk of recurrence of poor glycemic control in the future.

The mechanisms responsible for the abnormalities in the vascular wall associated with long standing diabetes mellitus are incompletely understood. The aim of this investigation was to assess the effects of angiotensin II and high glucose on the production of platelet-derived growth factor (PDGF) in human endothelial cells. For this purpose, a primary culture was obtained from fresh human umbilical cords by collagenase digestion of the vein interior. A high glucose medium increased the production of PDGF and a similar effect was observed by the addition of mannitol. These data are consistent with a stimulatory effect of glucose on PDGF that is mediated by the osmotic effect of this substance. Angiotensin II significantly increased PDGF in human endothelial cells and the effect was accompanied by a transient increase in cytosolic calcium. The angiotensin II-induced intracellular Ca2+ increases, PDGF production were completely abolished by saralasin and neomycin, respectively. We postulate that the increased production of PDGF by the vascular endothelium in response to high glucose and angiotensin II may participate in the development of the diabetic angiopathy.

Diabetic retinopathy is known to frequently deteriorate during pregnancy but the cause remains obscure. Vascular endothelial growth factor (VEGF), also known as vascular permeability factor (VPF), is a potent vascular endothelial cell mitogen which is mainly up-regulated by hypoxia, and is closely associated with the development and progression of diabetic retinopathy. To examine the influence of the drastic hormonal alterations during pregnancy on the worsening of diabetic retinopathy, we examined the effects of estradiol (E2) and progesterone (P4) on the production of VEGF/VPF in bovine retinal pigment epithelial cells in culture. The VEGF/VPF production was significantly elevated (214.5 +/- 28.3 ng/g protein, P0.01) by 48 h of exposure to a high concentration of P4(10 microM), which is still within the physiological range during pregnancy, compared to that of the control group (147.7 +/- 17.9 ng/g protein). However, E2 significantly stimulated the production of VEGF/VPF only at concentrations (100 microM) much higher than normally encountered during pregnancy. These two hormones were not observed to have a synergistic effect, at least at physiological concentrations. As the increase in serum P4 levels during pregnancy is reported to be greater in pregnant diabetic patients with progressive retinopathy, our findings suggest that P4 may contribute to the worsening of diabetic retinopathy during pregnancy by up-regulating intraocular VEGF levels.

The regulation of the production of platelet-derived growth factor (PDGF) and the influence of high glucose concentration, eicosapentaenoic acid (EPA) were studied in cultured human umbilical vein endothelial cells (HUE). The PDGF production of HUE increased markedly depending on glucose concentration. However, EPA (3 x 10(-4)M) markedly inhibited PDGF production [27.5 mM glucose group: 123 +/- 3% of control (5.5 mM glucose group), 27.5 mM glucose+EPA group: 104 +/- 5% of control]. These results suggested that a high glucose concentration and a high osmotic pressure-induced increase in PDGF production is involved in the development and progression of diabetic macroangiopathy. As eicosapentaenoic acid inhibits the PDGF production induced by high glucose concentration in HUE, use of this agent may exhibit anti-arteriosclerotic effects.

It has been demonstrated that activation of aldose reductase (AR; EC 1.1.1.21) in diabetic tissues plays an important role in the pathogenesis of diabetic complications. In the present study, the effects of non-enzymatic glycation of recombinant human AR (rhAR) on enzyme activity and affinity for its substrate (glyceraldehyde), co-factor (NADPH) and inhibitors (ARI; Sorbinil, Tolrestat, AL-1576 and Statil) were examined. Although rhAR was successfully nonenzymatically glycated with HPLC-purified [ 3 H]d-glucose, the Michaelis constant ( K m ) and catalytic efficiency (KcatKm) for glyceraldehyde, the K m for NADPH and the inhibitor constant ( K i ) for ARI did not change. These results suggest that the mechanism of AR activation and its insensitivity to inhibition observed in diabetic tissues cannot be attributed to its non-enzymatic glycation.

We produced an improved microembolism model of cerebral focal ischemia by injection of 1000–2000 microspheres (50 ± 5 μ m diameter) via a tube retrogradely inserted into the right external carotid artery in freely moving rats. The group injected with 2000 spheres showed a much more severe mortality rate as well as neurological signs than did the 1000-sphere group. Brain water content of the 2000-sphere group was examined and found to show an increase from 4 to 24 h after embolization in the right hemisphere, indicating serious brain edema. Severe neurological signs and individual deaths by embolization were most likely related to the extent of development of brain edema. Examination of learning behavior by shuttle-box avoidance revealed partial but significant impairment of learning in the 1000-sphere group. Autoradiographic studies for muscarinic acetylcholine receptors and protein kinase C binding sites were conducted. Both these binding sites decreased in number, but protein kinase C seems to be more susceptible to ischemic injury than muscarinic acetylcholine receptors. The observation was considered to be closely related with an impairment of learning. The present study suggests that our microembolism model in freely moving rats is useful for investigations of the early phase and late phase of cerebral ischemia.

To determine simultaneously the relative molecular mass (M(r)) of a native oligomeric protein, and the number and M(r) of its subunits, a method using two-dimensional electrophoresis was developed. To determine the M(r) of a native oligomeric protein, pore gradient gel electrophoresis was performed for the first dimension. Native proteins were dissociated into their subunits by sodium dodecyl sulphate (SDS) in a gel slice, then applied to SDS polyacrylamide gel electrophoresis for the second dimension to determine the M(r) of subunits. The advantage, accuracy, limitations and application of the method are discussed.

The relationship between glycation of the aortic elastin and calcium deposits in the aorta was studied in streptozocin (STZ)-induced diabetic rats. 5-Hydroxymethylfurfural (5-HMF) which was released from aortic elastin by acid, was assayed after STZ treatment as an index of early stage glycation. The amount of released 5-HMF increased at 5 weeks and paradoxically decreased at 10 weeks after STZ treatment, though it remained higher than that of control rats. This paradoxical pattern was reproduced by the in vitro incubation of elastin with glucose and it is presumably due to further advancement of glycation reactions in diabetic rats. The level of 5-HMF did not change significantly in control rats at corresponding time points of 9, 11 and 16 weeks of age. Fluorescence of porcine pancreatic elastase I-digested elastin which served as an index of advanced glycation, increased by 1.6 times at 3 weeks and reached a maximum of 1.9-fold higher than that of control rats at 10 weeks. The calcium content of the aorta at 10 weeks in diabetic rats was significantly increased by 1.4-fold compared with control rats. This study showed that the increased elastin glycation in the aorta even at the early stage of diabetes is associated with calcium deposit in the aorta. These results are consistent with the interpretation that elastin glycation in the aorta is the potential accelerating factor for diabetic macroangiopathy.

We investigated the effects of high glucose and hyperosmolality on platelet-derived growth factor (PDGF) production and PDGF-B chain mRNA levels in cultured human umbilical vein endothelial cells. Under an excess of ambient glucose (13.8 and 27.5mM) and a hyperosmolar condition (22.0mOsm/L with mannitol), PDGF concentrations in the culture medium were both significantly increased (10.3 ± 6.0%, 36.2 ± 7.2%, 48.5 ± 9.0% increase respectively compared with 5.5mM glucose). Parallel to protein secretion levels, PDGF-B chain mRNA levels showed a significant increase (57.7%, 103.7%, 210.8% increase), while no change of β-actin mRNA levels was observed. Thus, elevated PDGF released from endothelium by high glucose may play an important role in the pathogenesis of diabetic angiopathy.

It is known that Na-K,adenosine triphosphatase (ATPase) in cell membranes represents an important consumer of cellular energy, eg, adenosine triphosphate (ATP), and that the concentration and activity of this enzyme change in a dose-dependent manner with serum thyroid hormone levels. To examine the hypothesis that low triiodothyronine (T3) syndrome represents a cellular adaptation in generalized severe illnesses that saves tissue energy expenditure, we measured the muscle Na-K,ATPase concentration and its activity in rats that led to low T3 syndrome induced by fasting. The Na-K,ATPase concentration was measured by 3H-ouabain binding to soleus muscle, and its activity was measured by 42K uptake in the contralateral soleus muscle. The effects of refeeding or T3 administration on Na-K,ATPase in soleus muscle in fasted rats were also examined. Na-K,ATPase concentration and activity were both increased in hyperthyroid rats and decreased in hypothyroid rats. In the fasting state, they were decreased to as low as the levels seen in hypothyroidism. Furthermore, with fasting + refeeding or fasting + T3 administration, Na-K,ATPase in soleus muscle returned to the normal level. These results suggest that tissue energy expenditure, as assessed by Na-K,ATPase, in skeletal muscles of fasted rats with low T3 syndrome is actually decreased to levels seen in hypothyroidism, due at least partly to the decrease in serum T3 concentrations, and that there exist some adaptation mechanisms in the peripheral tissues for the accommodation of energy metabolism in the body through decreased thyroxine (T4) to T3 conversion.

Changes of glucose and lipid metabolism in NIDDM hypertensive patients during treatment with a new dihydropyridine derivative, nilvadipine, were examined by a randomized, crossover study comparing the results with those elicited by captopril in 18 patients for 12 weeks each. Nilvadipine (8 mg per day) and captopril retard (75 mg per day) caused a sufficient decrease in blood pressure without changing the pulse rate. Nilvadipine and captopril did not significantly change fasting plasma glucose, hemoglobin A1c, serum cholesterol, triglycerides, high-density lipoprotein cholesterol or apoprotein A-I, A-II and B levels in either of the 12-week treatments. In 75-g oral glucose tolerance tests carried out three times in each patient (before treatment and after 12 weeks of treatment with each drug), changes in plasma glucose and serum insulin levels were not significantly different among the three tests. These results demonstrate that nilvadipine as well as captopril are antihypertensive drugs without adverse effects on glucose and lipid metabolism in hypertensive patients with NIDDM.

The interaction of three incretin candidates, glucagon-like peptide-1(7–36)amide (t-GLP-1), gastric inhibitory polypeptide (GIP), and sulfated COOH-terminal octapeptide of cholecystokinin (CCK-8-S), on insulin and glucagon release from the isolated perfused rat pancreas was studied. Under the perfusate condition of 8.3 mmol/L glucose, coinfusion of 0.1 nmol/L t-GLP-1 and 0.1 nmol/L GIP resulted in an augmented insulin release greater than that obtained by the same dose of each peptide alone. The degree of stimulation elicited by t-GLP-1 and GIP reached a plateau at 0.3 nmol/L for both infusates, and no cooperative effect was observed by coinfusion at 0.3 nmol/L. Coinfusion of 0.1 nmol/L t-GLP-1 and 0.1 nmol/L CCK-8-S also resulted in an augmented insulin release greater than that obtained by the same dose of each peptide alone. A similar cooperative effect was observed by coinfusion at 0.3 nmol/L, 1 nmol/L, and 3 nmol/L. With the same perfusion experiments, glucagon release was not significantly affected by any peptide at concentrations of 0.1, 0.3, 1, or 3 nmol/L. The coinfusion of 1 nmol/L t-GLP-1 and GIP elicited a transient, but significant, increase in glucagon release. A similar result was obtained by the coinfusion of 0.3 nmol/L and 3 nmol/L t-GLP-1 and GIP, respectively. The coinfusion of t-GLP-1 and CCK-8-S did not affect the glucagon release. These results suggest that (1) t-GLP-1 and GIP cooperatively affect insulin and glucagon release at the nearly physiologic postprandial plasma concentrations of both peptides, and (2) t-GLP-1 and CCK-8-S cooperatively affect insulin release in a wide range of concentrations, although the physiologic significance of this interaction should be slight, considering the physiologic postprandial plasma concentration of CCK-8-S.

The role of His42, His188, and Lys263 residues in the catalytic action of human aldose reductase was investigated in association with various inhibitors of this enzyme by site-directed mutagenesis. While mutations at His42- greater than Gln, His42- greater than Tyr, His188- greater than Gln, and His188- greater than Tyr brought small change in the kinetic parameters, Lys263- greater than Glu mutation markedly increased the Km value for the substrate DL-glyceraldehyde by a factor of 60. Lys263- greater than Met substitution resulted in approximately 14 fold elevation of Km for the substrate. By contrast, mutation of Lys263- greater than Arg significantly decreased the Km for the substrate with concomitant reduction in kcat. Moderate increase in Km values for the cofactor NADPH was demonstrated for mutated enzymes. These results are indicative of the possible role of Lys263 in the substrate binding through electrostatic interaction. The inhibitor constants (Ki) for structurally diverse aldose reductase inhibitors against mutated enzymes demonstrated different degree of alteration, indicating binding sites of aldose reductase inhibitors on the enzyme molecule vary from one another, and some of the sites are more closely correlated with the physicochemical property of Lys263.

A peptide mitogen bombesin, which activates the phospholipase C-protein kinase C signaling pathway, induces a mepacrine-sensitive, dose-dependent increase in the release of [3H]arachidonic acid and its metabolites ([3H]AA) from prelabeled Swiss 3T3 fibroblasts. The effect is temporally composed of two phases, i.e. an initial transient burst that is essentially independent of extracellular Ca2+, and a following sustained phase that is absolutely dependent on the extracellular Ca2+. The initial transient [3H]AA liberation occurs concomitantly with bombesin-induced 45Ca efflux from prelabeled cells: both responses being substantially attenuated by loading cells with a Ca2+ chelator quin2. However, bombesin-induced intracellular Ca2+ mobilization by itself is not sufficient as a signal for the initial transient [3H]AA liberation, since A23187 potently stimulates 45Ca efflux to an extent comparable to bombesin but fails to induce [3H]AA release in the absence of extracellular Ca2+. The second sustained phase of the bombesin-induced [3H]AA release is abolished by reducing extracellular Ca2+ to 0.03 mM, although bombesin effects on phospholipase C and protein kinase C activation are barely affected by the same procedure. A protein kinase C activator phorbol 12,13-dibutyrate induces an extracellular Ca(2+)-dependent, slowly developing sustained increase in [3H]AA release, and markedly potentiates both phases of bombesin-induced [3H]AA release. Down-regulation of cellular protein kinase C completely abolishes all of the effects of phorbol dibutyrate, and partially inhibits the second but not the first phase of bombesin-induced [3H]AA release. These results indicate that bombesin-induced receptor-mediated activation of phospholipase A2 involves multiple mechanisms, including intracellular Ca2+ mobilization for the first phase, protein kinase C activation plus Ca2+ influx for the second phase, and as yet unknown mechanism(s) independent of intracellular Ca2+ mobilization or protein kinase C for both of the phases.

There are no definite guidelines in the treatment of non-insulin-dependent diabetes mellitus (NIDDM) as to whether the treatment of choice is insulin, a sulfonylurea or a combination of insulin and sulfonylurea. We have therefore tried to evaluate the long-term effects of these treatments on β-cell function in a rat model of NIDDM. NIDDM rats were prepared by the injection of streptozotocin (60 mg, i.p.) on the 5th day after birth. At 10 weeks, an oral glucose tolerance test (2 g/kg) was performed and rats were divided into 4 groups, each of which had the same mean glucose tolerance. The treatment of each group with either NPH insulin (4 U/kg/day), or oral gliclazide (10 mg/kg/day by a stomach cannula), or a combination of the above two, or a control (vehicle for gliclazide) was started from 12 weeks of age and continued for 6 months. Rats were fed ad libitum with standard rat chow. The weight gain of diabetic rats treated with gliclazide alone and of the vehicle-treated diabetic rats during 6 months was less than that of the other groups receiving insulin. The fasting plasma glucose of the insulin-only treated group stayed at the initial level for 6 months, but that of the other groups increased gradually. The frequency of deterioration of glucose tolerance for oral glucose loading (2 g/kg) in the insulin-only treated group was smaller than that in the other diabetic groups at 3 at 6 months after the start of treatment. The increase in plasma IRI after the oral glucose loading of the insulin-only treated group was the largest among the 4 groups at 6 months. In the pancreas perfusion experiment, the insulin response to glucose in the insulin-only treated group was more preserved than that in the other groups of diabetic rats after 6 months of treatment. These results suggest that treatment with insulin is effective in preserving β-cell function in a rat model of NIDDM, whereas treatment with a sulfonylurea agent is not only ineffective but might negate the protective effect of insulin because the insulin-plus-gliclazide treated group elicited results similar to those of the gliclazide-only treated group except for the weight gain.

Increased levels of platelet-derived growth factor (PDGF) may play a central role in the development of arteriosclerosis, but the factors that inhibit PDGF production in vascular endothelial cells remain mostly unknown. We examined the effects of cilostazol, an antithrombotic agent, and high glucose on PDGF production in cultured human umbilical vein endothelial cells (HUVEC). HUVEC grown in high glucose exhibits increased PDGF production, which was markedly inhibited by cilostazol. Since cilostazol inhibits PDGF production in HUVEC, its use may exhibit anti-arteriosclerotic effects in diabetic patients.

The effect of insulin on the transport of proline has been studied in cultured fibroblasts from normal individuals, non-insulin-dependent diabetic patients, and patients with Werner syndrome. In fibroblasts from normal individuals and those with diabetes mellitus, incubation with 10(-7) M insulin resulted in more than a twofold increase in the transport rate after about 14 h incubation. In contrast, fibroblasts from patients with Werner syndrome had a markedly attenuated response to insulin, suggesting a defect in insulin action on the transport of this amino acid in Werner syndrome.

The antagonistic effects of [D-Phe 25 ]gastrin-releasing peptide (GRP)(18–27) and [D-Arg 1 ,D-Pro 2 ,D-Trp 7,9 ,Leu 11 ]substance P (SP) on the stimulation of insulin release by GRP(18–27) from isolated canine pancreas were compared with that of [Ala 23 ]GRP(18–27). The stimulation of insulin release by 1 nM GRP(18–27) was reduced to 24.1% and 15.4% by the prior infusion of 1 μM of [D-Arg 1 ,D-Pro 2 ,D-Try 7,9 ,Leu 11 ]SP and 10 μM of [D-Phe 25 ]GRP(18–27), respectively. Glucagon release by GRP(18–27) was not affected by these peptides using the above concentrations. The results indicate that these peptides are antagonists of bombesin-like peptide receptors on pancreatic B-cells, although the inhibitory activities are lower than that of [Ala 23 ]GRP(18–27).

To evaluate the therapeutic efficacy of oral angiotensin-converting enzyme inhibition with low-dose (average 30 mg/day) captopril in diuretic-resistant hypertension, its long-term cardiocirculatory action was determined by dye-dilution method and venous-occlusion forearm plethysmography in 11 uncontrolled patients taking a thiazide diuretic. Significant declines in mean blood pressure (average 12.4 +/- 1.4 percent) and systemic vascular resistance (28.7 +/- 3.2 percent) accompanied an increase in cardiac output (24.8 +/- 4.1 percent). Forearm vascular resistance (16.0 +/- 2.7 percent) decreased considerably, but the decrease in limb vascular resistance did not parallel the fall in systemic vascular resistance in magnitude (p less than 0.01), indicating that arteriolar dilatation occurred on a selective basis. Plasma renin activity increased after therapy as plasma aldosterone levels consistently fell, while plasma norepinephrine concentrations were not changed. There was a direct correlation between pretreatment plasma renin activity and the magnitude of the decline in systemic vascular resistance (p less than 0.05). These findings suggest that the inhibition of angiotensin-converting enzyme with captopril in diuretic-resistant hypertensive patients improves cardiocirculatory function through selective dilatation. The reordering of regional blood flow, which appears to result from release of angiotensin-mediated vasoconstriction as well as the suppression of aldosterone, may underlie the prolonged benefit observed in these patients. This oral vasodilator in very low dose appears to represent an effective adjunct for the treatment of hypertension refractory to diuretics.

To clarify the independent roles of prostaglandins and the renin-angiotensin system in the pressor resistance to angiotensin II in Bartter's syndrome, the pressor responsiveness to exogenous angiotensin II was investigated in three patients with the syndrome during the administration of indomethacin synthesis, and captopril is an angiotensin-converting enzyme inhibitor. All the patients showed high plasma renin activity, increased urinary excretion of prostaglandin E, and pressor resistance of angiotensin II. An analogue of angiotensin II that had weak agonistic properties induced a marked fall in blood pressure. Pretreatment with indomethacin (150 mg/day) decreased baseline plasma renin activity and reversed the hypotensive effect of the analogue of angiotensin II. Apparently, our data support the concept that pressor resistance ultimately results from the increase in the concentration of endogenous angiotensin II. However, the augmentation of indomethacin was significantly (p less than 0.01) greater in magnitude than the response obtained with captopril, although the concentration of plasma angiotensin II prior to each infusion of angiotensin II was the same. This observation could be explained by the finding that indomethacin suppressed both systems, but captopril inhibited only the renin-angiotensin system. Evidence presented herein suggests that the abnormalities in the vascular reactivity to angiotensin II may result from, not only the decreased number of receptor sites as a results of the increased concentration of endogenous angiotensin II, but also from the alteration of the end-organ sensitivity to angiotensin II via overproduction of prostaglandins.

A neuropeptide, substance P (1–50 μM) caused a prompt but transient rise in tissue cyclic AMP levels and also increased the release of thyroid hormones from canine thyroid slices. While norepinephrine markedly inhibited the stimulation by TSH of such paramaters as reported previously, substance P had no effect. These results suggest that substance P may play a regulatory role in thyroid gland functions in a manner different from norepinephrine.

Daily intake of 8.0 g per day of fructo-oligosaccharides for fourteen days significantly reduced mean fasting blood glucose levels by 15 mg/dl, mean serum total cholesterol levels by 19 mg/dl and LDL-cholesterol levels by 17 mg/dl in diabetic subjects (n=18), while the control diabetic subjects (n=10) who were given 5.0 g per day of sucrose showed no significant changes. The levels of serum HDL-cholesterol, triglycerides or free fatty acids were not significantly affected either by fructo-oligosaccharides or sucrose. These results indicate that the daily intake of fructo-oligosaccharides ameliorates the derangements of carbohydrate and lipid metabolism in diabetic subjects.

The β-adrenergic receptor-coupled adenylate cyclase system has been investigated in normal and Werner's syndrome fibroblasts. The basal levels of cAMP in Werner and normal control cells were similar, whereas the isoproterenol-induced increase in cAMP levels was far less for Werner cells than for control cells. In the broken cell preparations isoproterenol stimulated the adenylate cyclase of only control cells, not of Werner cells, although NaF or prostaglandin E 1 stimulated the enzyme of both cells to the same extent. The β-adrenergic receptor concentrations analyzed with hydrophilic radioligand were nearly equal in Werner and in control cells. A reduction of functional activity of the β-adrenergic receptor in Werner cells is thus suggested.

Thyroid stimulating hormone (TSH) increased cyclic AMP levels approximately 10–20 fold in canine thyroid slices after 30 min incubation. Thereafter the cyclic AMP level declined reaching about 50% of the maximal by 90 min even in the presence of 10 mM theophylline. When phentolamine, an α-adrenergic blocker, was added with TSH to the incubation medium, the decline of cyclic AMP levels that followed the peak was markedly diminished. The maximal effect of phentolamine was observed at a concentration of 10−6M. A similar decline of the cyclic AMP levels after the peak was observed when the tissues was stimulated by prostaglandin E1 or cholera toxin and the decline was again prevented by phentolamine. Phentolamine alone had no significant effect on the basal cyclic AMP levels. Phenylephrine, an α-adrenergic agonist, diminished the rise of cyclic AMP levels induced by TSH. Norephinephrine, a physiologic adrenergic stimulator, caused a marked inhibition of the elevation of cyclic AMP levels induced by prostaglandin E1 or cholera toxin as was the case by TSH (Life Sciences 21, 607, 1977). The norepinephrine effect was abolished by phentolamine, but not by propranolol, a β-adrenergic blocker. These results indicate that α-adrenergic actions may be involved in the counter-regulation of cyclic AMP levels in canine thyroid glands.

Treatment of bovine thyroid plasma membranes with phospholipase A or C inhibited the stimulation of adenylate cyclase activity by thyroid-stimulating hormone (TSH). In general, basal and NaF-stimulated adenylate cyclase activity was not influenced by such treatment. When plasma membranes were incubated with 1–2 units/ml phospholipase A, subsequent addition of phosphatidylcholine or phosphatidylserine but not phosphatidylethanolamine partially restored TSH stimulation. Phosphatidylcholine was more effective than phosphatidylserine in that it caused greater restoration of the TSH response and smaller amounts of phosphatidylcholine were active. However, when the TSH effect was obliterated by treatment of plasma membranes with 10 units/ml phospholipase A, phospholipids were unable to restore any response to TSH. Lubrol PX, a nonionic detergent, inhibited basal, TSH- and NaF-stimulated adenylate cyclase activities in thyroid plasma membranes. Although phosphatidylcholine partially restored TSH stimulation of adenylate cyclase activity in the presence of Lubrol PX, it did not have a similar effect on the stimulation induced by NaF. These results indicate that phospholipids are probably essential components in the system by which TSH stimulates adenylate cyclase activity in thyroid plasma membranes. The effects do not seem to involve the catalytic activity of adenylate cyclase but the data do not permit a distinction between decreased binding of TSH to its receptor or impairment of the signal from the bound hormone to the enzyme activity.

Adenyl cyclase activity was assayed in a plasma membrane fraction prepared from bovine thyroid using sucrose gradients. Its specific activity was ten times greater than that of the original homogenate. The activity in the plasma membrane fraction was stimulated five-fold by TSH as compared to only a two-fold increase in the whole homogenate. Electron microscopy of the membrane fraction indicated that it was contaminated by only a minimum amount of other cellular components. The Na + -K + -activated ATPase in this fraction had a specific activity ten times that in the original homogenate. The membrane fraction was devoid of cytochrome C oxidase but did contain higher specific activity DPNH-cytochrome C reductase than the original homogenate.

Substitution of K + or choline for Na + in Krebs-Ringer bicarbonate buffer increased basal glucose oxidation and 3 H-adenine incorporation into 3 H-CAMP but had no effect on CAMP concentrations. In high K + buffer, TSH (3 mU/ml and 50 mU/ml) and prostaglandin E 1 (PGE 1 ) (0.1 μg/ml and 10 μg/ml) had little or no effect on glucose oxidation while their stimulation of 3 H-CAMP formation and CAMP levels was equivalent to that obtained using regular Krebs-Ringer bicarbonate buffer. These results indicated that high K + inhibited the effects of CAMP and not its generation. The increased basal glucose oxidation in high K + buffer probably reflected increased membrane permeability since slices incubated with 14 C-1-glucose and regular Krebs-Ringer bicarbonate buffer did not have augmented 14 CO 2 production when they were later transferred to high K + buffer. The increased 3 H-adenine incorporation into 3 H-CAMP cannot be explained by increased membrane permeability, but appeared to reflect an effect on the intracellular conversion of 3 H-adenine to 3 H-ATP or 3 H-ATP to 3 H-CAMP. The effect of high K + probably reflected low Na + in the buffer since similar results were obtained when choline chloride was substituted for NaCl in Krebs-Ringer bicarbonate buffer. Exclusion of Ca ++ from Krebs-Ringer bicarbonate buffer decreased basal glucose oxidation, but had no effect on 3 H-adenine conversion to 3 H-CAMP or CAMP concentrations. Although effects of TSH and PGE 1 on glucose oxidation were diminished in buffer devoid of Ca ++ , their stimulation of 3 H-CAMP formation and CAMP levels was similar in the presence and absence of Ca ++ . These results demonstrate that changes in the ionic composition of the buffer do not interfere with activation of the adenyl cyclase-CAMP system but modify some of the subsequent actions of the CAMP formed.

Cyclic guanosine 3',5'-monophosphate (cyclic GMP) levels were determined in dog thyroid slices using the protein-binding method. [3H]Guanine incorporation into cyclic GMP was also determined by thin layer chromatography. Basal levels of cyclic GMP in dog thyroid slices were usually 10 to 30 pmoles per g wet tissue. These values were at least 10-fold lower than the cyclic adenosine 3',5'-monophosphate (cyclic AMP) levels. Cyclic GMP levels were significantly increased by acetylcholine (5.5 x 10-7 m to 5.5 x 10-5 m) and NaF (10-2 m) after a 1-min incubation. Increases (5 to 10-fold) were observed after 5- and 20-min incubations with acetylcholine (5.5 x 10-5 m) and after a 20-min incubation with NaF (10-2 m). Neither thyroid-stimulating hormone (100 milliunits per ml) nor prostaglandin E1 (2.8 x 10-5 m) increased cyclic GMP levels during 1-,5-, and 20-min incubations. Acetylcholine and NaF also increased [3H]guanine incorporation into cyclic GMP. The effect of acetylcholine was inhibited by atropine (1.6 x 10-4 m). Acetylcholine and NaF had little or no effect on cyclic AMP levels, while thyroid-stimulating hormone and prostaglandin E1 markedly increased cyclic AMP concentrations. In liver slices of the rat, acetylcholine increased cyclic GMP levels 2-fold, but NaF had no effect. These results indicate that the levels of cyclic GMP and cyclic AMP in dog thyroid slices are regulated by separate hormonal and metabolic mechanisms.