Protein Kinase C- alpha/betaII, delta, and zeta/lambda involvement in ethanol-induced MAPK expression in vascular smooth muscle cells.

Protein Kinase C (PKC) exists as one of twelve serine/threonine isoforms and has been found to mediate ethanol-induced activation of the Mitogen-Activated Protein Kinase (MAPK) pathway. The aim of this study was to determine the PKC isoform(s) that are mediators of ethanol-induced MAPK activity (ERK 1 and 2) and to verify the necessity of calcium in this activation process using cell culture in the presence and absence of ethanol, and other agents that modulate PKC expression. Western blotting analysis was used to assess the effect of ethanol on activating classical (alpha/ssII), novel (delta) and atypical (zeta/lambda) PKC isoforms in vascular smooth muscle cells (VSMCs). The results indicate that ethanol treated VSMCs express the classical PKC-alpha/ssII, novel PKC-delta, and atypical PKC-zeta/lambda isoforms. The expression of PKC-alpha/ssII was inhibited within the first two min of stimulation, followed by activation with maximum expression at 10 min. Similarly, PKC-delta and zeta expressions were suppressed during the first two min of ethanol stimulation with maximum increase in expressions at 10 min. The PKC inhibitor GF109203X and the calcium chelating agent BAPTA, enhanced ethanol-induced PKC expression, whereas, diltiazem reduced expression of PKC by 10% of control. On the other hand, BAPTA in the presence of GF10203X inhibited expression of ERK 1 & 2 downstream from the PKC pathway, whereas, BAPTA alone enhanced expression. These results demonstrate also that classical, novel, and atypical PKCs respond to ethanol during the initial phase of activation of ERK 1 & 2.

Cross-talking between calcium and histamine in the expression of MAPKs in hypertensive vascular smooth muscle cells.

Histamine (HA) is one of many neurotransmitters that have been implicated in cardiovascular functioning. Alterations in vascular smooth muscle due to the effects of histamine have been suggested. We investigated the modulatory effect of HA on mitogen activated protein kinase (MAPK) expression, specifically extracellular regulating kinase (ERK) 1 & 2 in vascular smooth muscle cells (VSMCs) from both spontaneously hypertensive (SHR) and control Wistar Kyoto (WKY) rats. Cross-talking between calcium (Ca2+) and HA during HA-induced modulatory effect on MAPK expression in SHR VSMCs was also investigated. A stimulatory increase in expression of ERK 1 & 2 was observed to be dose and time dependent with maximum expression occurring within 5 min in both SHR and WKY VSMCs. The stimulatory increase in expression is persistent for 60 min in SHR VSMCs, whereas, in WKY cells the stimulatory effect persists for only 20 min. Mepyramine, the H1 receptor antagonist, reduced the HA-induced increase in ERK 1 & 2 significantly in SHR VSMCs. A reduction in the HA stimulated increase in ERK 1 & 2 expression was observed at 20 min of exposing cells to diltiazem, the calcium channel blocker, whereas, the calcium chelator, BAPTA effect on ERK 1 & 2 expression was observed within 5 min in SHR VSMCs. The data demonstrates that cross-talking occurs between HA stimulation and Ca2+ induction during HA-induced activation of ERK 1 & 2 in VSMCs of both cell types. Although both intracellular calcium ([Ca2+]i) and extracellular Ca2+ maybe involved in the activation of ERK 1 & 2 by HA, the dependence on [Ca2+]i is more dramatic than the dependence on extracellular Ca2+ in hypertensive cells, which may contribute to the role of HA as a risk factor of hypertension in VSMCs of the aorta.

Dietary supplementation with marine fish oil improves in vitro small artery endothelial function in hypercholesterolemic patients: a double-blind placebo-controlled study.

BACKGROUND: Marine fish oils improve vascular function, but the mechanism of benefit is unclear. We conducted a study to examine the effects of fish oils given to hypercholesterolemic patients on small artery function in vitro. METHODS AND RESULTS: In a randomized, double-blind, placebo-controlled trial, subcutaneous gluteal fat biopsies were taken from 16 hypercholesterolemic patients (serum total cholesterol, 7.97+/-0.16 mmol/L [mean+/-SEM]) and 12 age- and sex-matched control subjects (mean cholesterol, 5.11+/-0.34 mmol/L). Small arteries were mounted on a wire myograph for isometric tension experiments. Patients and control subjects were randomized to receive fish oil (Maxepa 5 capsules BID) or placebo for 3 months. A second biopsy was taken and the studies were repeated. Relaxation to acetylcholine was significantly improved in the hypercholesterolemic group given Maxepa but not in the placebo group (mean maximum relaxation before and after, 48+/-6.2% and 68.83+/-2.19%, P=.0054). The dysfunction was not restored to control values (84.3+/-5.2%, P=.0002). There was also a smaller but significant impairment in endothelium-independent relaxation provoked by sodium nitroprusside (P<.01). A good correlation between the increase in eicosapentanoic acid (n=3) in red cell membrane and improvement in relaxation in the hypercholesterolemic group given fish oils was seen (r=.781, P<.02). CONCLUSIONS: Marine fish oil significantly improved endothelial function in peripheral small arteries in hypercholesterolemia patients. This may provide a mechanism for the beneficial effects of these fatty acids in coronary heart disease.

Successful coronary pace-termination of ventricular tachycardia during coronary angioplasty.

Coronary pacing has been performed to treat bradycardias occurring during percutaneous transluminal coronary angioplasty (PTCA) using an angioplasty guidewire. We describe a case of a 62-year-old man who developed ventricular tachycardia (VT) during PTCA. The tachycardia was successfully terminated by overdrive pacing via an angioplasty guidewire.

Soluble P-selectin in hyperlipidaemia with and without symptomatic vascular disease: relationship with von Willebrand factor.

Soluble P-selectin (CD62P) may arise from platelets, the endothelium, or both, and raised levels are found in those with thrombotic disease and atherosclerosis. To determine whether these increased levels in atherosclerosis are related to hypercholesterolaemia, blood samples were obtained from 86 patients (43 with symptomatic vascular disease) attending a hypercholesterolaemia clinic, and 86 age- and sex-matched controls. Parallel measurement of endothelial cell product von Willebrand factor helped define the origin of sP-selectin. Using ELISAs, soluble P-selectin was higher (median 290 ng/ml, range 80-735, P < 0.05) in patients with vascular disease than in both patients with uncomplicated hypercholesterolaemia (median 210 ng/ml, range 55-550), and controls (median 190 ng/ml, range 48-500). Von Willebrand factor was raised in both patients with uncomplicated hypercholesterolaemia (115 +/- 26 IU/dl, P < 0.05) and patients with hypercholesterolaemia and vascular disease (129 +/- 32 IU/dl, P < 0.02) compared with controls (102 +/- 30 IU/dl). Levels of soluble P-selectin did not correlate with von Willebrand factor, total low-density-lipoprotein (LDL) or high-density-lipoprotein (HDL) cholesterol or triglycerides levels, blood pressure or smoking, but von Willebrand factor correlated with LDL cholesterol (r = 0.42, P < 0.05). We conclude that plasma lipoproteins are not a major influence on levels of soluble P-selectin.

In vitro responses of human peripheral small arteries in hypercholesterolemia and effects of therapy.

BACKGROUND: Studies in both animals and humans with raised lipid levels have demonstrated abnormalities in vascular function usually manifested by an impairment in endothelium-dependent vasorelaxation. This is believed to be an early event in atheroma formation. There are few data on the effects on vascular function in humans of lowering serum lipids. We conducted a study to investigate the effects of cholesterol reduction on the in vitro function of human peripheral small arteries in middle-aged patients with hypercholesterolemia. METHODS AND RESULTS: Subcutaneous gluteal fat biopsies were taken from 18 hypercholesterolemic (HC) patients (mean +/- SEM serum total cholesterol, 9.7 +/- 0.57 mmol/L) and 16 age- and sex-matched control subjects (mean cholesterol, 4.69 +/- 0.18 mmol/L). Subcutaneous small arteries (internal diameter, < 330 microns) were dissected and mounted on a wire myograph for isometric tension measurements. The HC patients showed impaired relaxation to acetylcholine (10(-9) to 10(-6) mol/L) after preconstriction with the thromboxane A2 analogue U46619 (10(-6) mol/L, mean maximum relaxation, 42.9 +/- 5.4%) compared with control subjects (85.7 +/- 4.0%, P < .00001). Incubation with the nitric oxide substrate L-arginine (3 mmol/L) improved the endothelium-dependent vasorelaxation response to acetylcholine (70.9 +/- 6.0%, P < .01) in patients but not in control subjects. Also, there was a smaller but significant difference in responses to the endothelium-independent agent sodium nitroprusside (10(-9) to 10(-6) mol/L) between the HC group (mean maximum relaxation, 76.9 +/- 6.0%) and the control subjects (89.7 +/- 6%; P < .01). A total of 10 patients had a second gluteal skin biopsy and repeat functional studies after successful cholesterol-lowering therapy after a mean period of 9.9 +/- 4.7 months. A significant reduction in total and LDL cholesterol was achieved (5.29 +/- 0.2 and 3.23 +/- 0.21 mmol/L, respectively; P < .001). This restored vasorelaxation to control values in response to both acetylcholine (mean maximum relaxation, 83.3 +/- 3.8%; P < .0001) and sodium nitroprusside (87.9 +/- 4.8%, P < .01). Although both groups were normotensive, there were significantly higher blood pressures in the HC group compared with control subjects (139 +/- 4.1 versus 123 +/- 3.0 mm Hg systolic, P < .01; 84 +/- 1.3 versus 75 +/- 2.2 mm Hg diastolic, P < .01). There was no difference in initial blood pressures between the entire group of 18 and the 10 patients who had repeat biopsies. The blood pressures fell to control values after cholesterol reduction (129.33 +/- 4.93 mm Hg systolic and 72.33 +/- 2.93 diastolic mm Hg, P < .02 relative to pretreatment values). CONCLUSIONS: These results demonstrate abnormalities of both endothelium-dependent and -independent relaxation in human peripheral small arteries that are normalized with effective lipid lowering. The changes in blood pressure may have been secondary to the improvement in vascular function.

Intense immunosuppression in chronic progressive multiple sclerosis: the Kaiser study.

The value of a short course of intensive immunosuppression with cyclophosphamide in stabilising chronic progressive multiple sclerosis (MS) was examined in a randomised single-blinded, placebo-controlled clinical trial. Forty two patients, from the Kaiser Permanente Medical Care Program, Northern California, were studied. Twenty two patients received a short course of cyclophosphamide in an outpatient neurology clinic until their leucocyte counts fell below 4000/mm3, and 20 patients received folic acid. Level of disability, impairment of functional systems, and performance of social roles were assessed before randomisation and reassessed 12, 18, and 24 months after therapy. In both the cyclophosphamide and folic acid groups, the mean level of disability increased from the baseline examination to the 12 month follow up examination (the primary endpoint) by 0.5 on Kurtzke's Expanded Disability Status Scale, indicating similar disease progression in the two groups. Although immunosuppression therapy can be safely administered to MS patients in an outpatient clinic, evidence of substantial benefits was not found.

Daytime polysomnogram diagnosis of sleep disorders.

The daytime polysomnogram was used to evaluate 310 consecutive patients with suspected sleep disorders, referred mainly because of excessive daytime sleepiness. Abnormalities consistent with pathologic sleep apnoea were present in 102 cases, and with narcolepsy-cataplexy in 49 cases. The daytime polysomnogram is a readily accessible, accurate, and cost-effective method for diagnosing many sleep disorders.

Botulism: a case with associated sensory abnormalities.

A 25-year-old man with type A botulism had prominent sensory symptoms and signs, which are findings that have not been previously reported to date. A sensory mononeuropathy multiplex was suspected. Sensory abnormalities do not exclude the diagnosis of botulism.

Projections from the brain stem reticular formation to laminae I and II of the spinal cord. Studies using light and electron microscopic techniques in the North American opossum.

The horseradish peroxidase and autoradiographic methods show that laminae I and outer II are innervated by the nucleus reticularis gigantocellularis pars ventralis, and the nucleus reticularis pontis pars ventralis. Both areas contain neurons of the indolamine type and probably account for the indolamine-like varicosities which are present within laminae I and II. Degeneration materiom the above nuclei end on small dendritic shafts and spines as well as on vesicle-filled proflies. The terminals identified formed asymmetrical contacts and contained clear as well as dense-cored vesicles. No terminals were present within glomeruli. A projection to laminae I and outer II also arises within the dorsolateral pons and several ines of evidence suggest that it is catecholaminergic. The electron microscope revealed that axons from the dorsolateral pons are fairly numerous within laminae I and II, but that terminal contacts are relatively rare. Those present are asymmetrical and alternate with intermediate-sized dendrites. They contain clumps of clear, spherical vesicles as well as larger vesicles with a variety of dense cores.

Observations on the development of brainstem-spinal systems in the North American oppossum.

The North American oppossum is born 12 to 13 days after conception and and is available for 90 days or more in an external pouch where it can be observed and experimentally manipulated. It is of particular interest that the hindlimbs of the newborn opossum are very immature and remain immobile for a week or more after birth. Degeneration techniques reveal that immature brainstem axons are present within the marginal zone of the lumbosacral cord before hindlimb movements begin (our state I) and material processed for formaldehyde induced fluorescence shows that some of them transport monoamines. Several lines of evidence suggest that part of the fluorescent axons arise within the nucleus locus coeruleus. At this early stage the electron microscope reveals that all brainstem-spinal axons are small (0.1--0.4 micrometer in diameter) and unmyelinated. By the time random hindlimb movements can be observed (stage II), brainstem axons, including those transporting monoamines, can be demonstrated to have grown into limited areas of the intermediate zone of the lumbosacral cord and to arise from most of the areas contributing to them in the adult animal (horseradish peroxidase technique). Such axons are still immature and it is not yet clear that they have formed synaptic terminals. Brainstem axons continue to grow into the intermediate zone of the lumbosacral cord for some time and come to occupy all of their adult territories before thoracic transection produces obvious change in hindlimb motility (beginning of stage III). It is still another 20 days or so before thoracic transection produces spinal shock comparable to that in the adult animal. The relatively mature use of the hindlimbs and the full expression of spinal shock correlate with changes in the technique and survival time needed to demonstrate degenerating brainstem axons in experimental material.

An electron microscopic study of rubrospinal projections to the lumbar spinal cord of the opossum.

The rubrospinal system is a major suprasegmental input to the important interneuronal pool at the base of the lumbar dorsal horn in the North American opossum. After appropriate lesions, rubral axons and their synaptic terminals were found in electron micrographs of lamina IV, V and VI as well as within the dorsal extreme of lamina VII. Degenerating terminals contact small diameter dendrites in the lateral terminal zone and large dendritic profiles in the medial terminal zone. Correlating these data with the dendritic arborizations of interneurons in Golgi preparations and with existing physiologic studies, it appears that interneurons in the intermediate and medial aspects of lamina V, VI and VII receive rubral input on both their proximal and distal dendrites.

Composition of the domestic water supply and the incidence of fractures and encephalopathy in patients on home dialysis.

Of the 202 patients undergoing home dialysis in the Trent region, 11 developed dialysis encephalopathy, 21 suffered spontaneous fractures, and 36 who had undergone dialysis for over four years had neither of these complications. Because the incidence of complications seemed to be unevenly distributed the water supplies were analysed. Water supplied to the homes of the patients with fractures or encephalopathy contained significantly less calcium and fluorine and significantly more aluminium and manganese than that piped to patients without these complications. The high aluminium concentrations in the bone of patients with encephalopathy was confirmed, but aluminium concentrations in the brains from three patients with encephalopathy were not increased. Patients who undergo dialysis in areas where water contains high aluminium concentrations should be supplied with deionisers.

Possible cobalt toxicity in maintenance hemodialysis patients after treatment with cobaltous chloride: a study of blood and tissue cobalt concentrations in normal subjects and patients with terminal and renal failure.

The myocardial cobalt concentration in a patient who died 3 months after treatment with cobalt was 25-80 times greater than the concentration in control samples. Blood cobalt concentrations in maintenance hemodialysis patients who had been treated 13-20 months previously with cobaltous chloride were significantly higher than those in maintenance hemodialysis patients who had not received cobalt. Prospective studies of blood cobalt concentrations in maintenance hemodialysis patients and normal subjects after the administration of cobaltous chloride were carried out. It was found that prolonged elevation of blood cobalt concentrations occurred in both normals and maintenance hemodialysis patients, but that the blood cobalt concentrations were much higher in the dialysis patients. The urinary excretion of cobalt following the administration of a single dose of cobaltous chloride was studied in two normal subjects. Cobalt metabolism and toxicity are discussed. In view of the limited therapeutic gains to be expected and because of the lack of information regarding the long term significance of elevated blood cobalt concentrations, it is concluded that cobalt should not be used in the treatment of the anemia of patients with sever renal failure.

Origin, course and termination of corticospinal fibers in a prosimian primate, Galago.

This report describes the origin, course, and termination of cortical projections to the spinal cord in the bushbaby, Galago, Although the position and extent of these projections are similar to those reported in other primates, there is also (1) a somatotopic organization present in the motor-sensory cortex and in its projections to the spinal cord, and (2) additional nuclei in the medial base of the dorsal and ventral horn which receive afferents from the motor-sensory amalgam. This medial organization may be related to the innervation of axial musculature in Galago, an animal displaying a vertical clinging and leaping locomotive behavior.