K252a inhibits proliferation of glioma cells by blocking platelet-derived growth factor signal transduction.

Growth factors are known to regulate glioma proliferation. The glioma cell lines U87 and T98G were examined for evidence of an autocrine stimulatory loop involving the neurotrophin family of growth factors. Although neurotrophin-3 and TrkC RNA were detected by reverse transcription-PCR, there was no evidence of significant interaction between neurotrophin-3 and its cognate receptor TrkC. The microbial alkaloid K252a has been described to inhibit both Trk tyrosine kinase activity and neuroblastoma cell proliferation. K252a inhibited proliferation in U87 (IC50 = 1170 nM) and T98G (IC50 = 529 nM) but induced apoptosis in U87 cells only. At concentrations of 500 nM to 1 microM, K252a blocked only platelet-derived growth factor (PDGF)-mediated receptor autophosphorylation. These results suggest that an autocrine loop involving PDGF is functional and important for maintaining tumor growth. There is no evidence to support the existence of a neurotrophin-mediated autocrine loop. K252a, through inhibition of PDGF signal transduction, may be a novel therapeutic agent in the treatment of human gliomas.

Adenosine diphosphate potentiates the inhibition of norepinephrine-induced relaxation by 5-hydroxytryptamine in canine coronary arteries.

Vasoactive substances released from aggregating platelets inhibit beta-adrenergic neurotransmission in coronary arteries. Studies were carried out on the effects of two such vasoactive substances on canine coronary arteries, at concentrations equivalent to that released by platelets under physiological conditions. 5-Hydroxytryptamine (5 X 10(-7) M) reduced the sensitivity of coronary artery ring segments to the beta-adrenergic relaxing effects of norepinephrine. Adenosine diphosphate (3 X 10(-6) M) further reduced the sensitivity to norepinephrine caused by 5-hydroxytryptamine, while the nucleotide alone had no significant effect. 5-Hydroxytryptamine and adenosine diphosphate acted selectively on the norepinephrine-induced relaxation; whereas the relaxatory response of the vessel to nitroprusside, a direct muscle relaxant, was unaffected. 5-Hydroxytryptamine caused contraction of the tissue, but this opposing response did not account for the inhibition of the norepinephrine-induced relaxation observed in the presence of the indoleamine. The decreases in sensitivity to norepinephrine caused by 5-hydroxytryptamine and adenosine diphosphate were prevented by the serotonin receptor antagonist, methiothepin. The potentiation by adenosine diphosphate of the 5-hydroxytryptamine-induced shift in the relaxation caused by norepinephrine was blocked by the purine receptor antagonist, 8-(p-sulfophenyl)-theophylline. Neither adenosine nor alpha,beta-methylene adenosine diphosphate potentiated the action of 5-hydroxytryptamine, suggesting that phosphate hydrolysis of the nucleotide is required for the action of adenosine diphosphate. These results suggest that adenosine diphosphate potentiates the inhibitory effect of 5-hydroxytryptamine on the beta-adrenergic response of coronary arteries exposed to vasoactive substances released from platelets.

Intraarterial papaverine infusion for cerebral vasospasm after subarachnoid hemorrhage.

PURPOSE: To evaluate the techniques and efficacy of intracranial intraarterial papaverine infusion for symptomatic vasospasm after subarachnoid hemorrhage caused by aneurysm rupture. METHODS: Papaverine was infused on 19 occasions in 14 patients, 6 hours to 2 days after spasm became apparent clinically. Sixty vascular territories were treated. Infusion was made into the supraclinoid internal carotid artery 20 times, cavernous internal carotid artery once, selective A1 anterior cerebral artery 8 times, M1 middle cerebral artery 7 times, and basilar artery 3 times. Papaverine doses ranged from 150 to 600 mg and exceeded 400 mg on 8 occasions. RESULTS: Angiographic improvement occurred in 18 (95%) of the 19 treatment sessions: results were excellent in 3 sessions, moderate in 8, and mild in 7. The best angiographic results often were obtained with superselective infusion, although angiographic results did not always correlate with clinical response. Seven (50%) of the 14 treated patients showed dramatic acute clinical improvement within 24 hours of papaverine therapy, and there was no clinical evidence of recurrent vasospasm in these patients. Recurrence of angiographic vasoconstriction was demonstrated in three patients; one showed marked clinical improvement after a second treatment. There were no episodes of systemic hypotension in any of the cases. Monocular blindness developed in one patient because of papaverine infusion near the ophthalmic artery. CONCLUSIONS: Papaverine was effective in dilating narrowed arteries in most patients with symptomatic vasospasm caused by subarachnoid hemorrhage. This series showed encouraging clinical results with no recurrence of neurologic deterioration in those patients who responded well to papaverine. Superselective infusion appears to be indicated in some cases for adequate papaverine delivery.

Accumulation of 5-hydroxytryptamine leads to dysfunction of adrenergic nerves in canine coronary artery following intimal damage in vivo.

Previous in vitro studies have demonstrated that coronary artery adrenergic nerves are a principal site of accumulation of 5-hydroxytryptamine released from aggregating platelets. The purpose of this study was to determine whether 5-hydroxytryptamine is accumulated by adrenergic nerves at sites of endothelial damage and platelet aggregation in vivo. Coronary artery 5-hydroxytryptamine content and response to in vitro adrenergic nerve stimulation were studied in dogs 24 hours following balloon catheter-induced intimal injury. 5-Hydroxytryptamine content was significantly increased in the catheter-damaged arteries, and there was a coincident decrease in the content of norepinephrine. The relaxation caused by acetylcholine was abolished in the catheter-injured arteries, indicating loss of this endothelial cell-mediated function. The normal beta-adrenergic relaxation caused by nerve stimulation was inhibited, and in some cases, contractions resulted; these effects were prevented by serotonergic receptor antagonists. The sensitivity to exogenously added norepinephrine was unchanged, indicating that the changes in the response to nerve stimulation were not due to an altered smooth muscle response to the native neurotransmitter. These observations indicate that following intimal damage, which produces platelet aggregation on the luminal surface of the blood vessel, 5-hydroxytryptamine can assume a transmitter role in coronary artery adrenergic nerves and thereby cause their dysfunction.

Loss of selective endothelial cell vasoactive functions caused by hypercholesterolemia in pig coronary arteries.

The influence of hypercholesterolemia on the reactivity of coronary arteries was investigated after feeding a high-cholesterol diet to pigs for 9 weeks. After this duration of hypercholesterolemia, the fatty or intimal proliferative changes of atherosclerosis were not yet evident in the coronary arteries by light or electron microscopy. Changes in isometric tension were compared in isolated ring segments of coronary arteries from normal and hypercholesterolemic animals. The endothelium failed to inhibit contractions caused by 5-hydroxytryptamine in coronary arteries from hypercholesterolemic animals, but it did so in normal vessels. In contracted arteries, endothelium-dependent relaxations caused by 5-hydroxytryptamine and substance P were reduced by hypercholesterolemia. In contrast, endothelium-dependent relaxations mediated by norepinephrine acting at alpha 2-adrenoceptors and those caused by the calcium ionophore A23187 were unaffected. Endothelium-independent beta-adrenergic relaxations caused by norepinephrine, as well as those caused by nitroprusside, and papaverine also were unaffected by hypercholesterolemia. The loss of selective endothelial cell receptor-mediated relaxation suggests that it is not the ability of the coronary artery endothelium to elaborate vasodilators, but the initiation of the coronary artery endothelial cell response to 5-hydroxytryptamine and substance P that is affected by hypercholesterolemia. Thus, during hypercholesterolemia, selective endothelial cell dysfunction giving rise to abnormal coronary artery reactivity precedes the onset of coronary artery atherosclerosis.

Adrenergic denervation in rabbits with diabetes mellitus.

The influence of alloxan-induced diabetes mellitus on the sympathetic neuroeffector junction of the rabbit carotid artery denuded of endothelium was studied. Six weeks of diabetes resulted in a neuropathy characterized by a 38% reduction in the arterial content of norepinephrine. Norepinephrine release from the nerves measured from electrically stimulated superfused arterial segments was decreased. The cocaine-sensitive accumulation of [3H]-norepinephrine (NE) was also reduced, reflecting decreased neuronal uptake. The consequences of these prejunctional changes were studied by measuring isometric contractions of arterial rings caused by electrical nerve stimulation or by exogenous norepinephrine. Despite the reduced release of norepinephrine, neurogenic contractions were normal, suggesting an increased sensitivity of the smooth muscle. After neuronal uptake was blocked, the neurogenic contractions of diabetic arteries were less than normal, reflecting the reduction in transmitter release. The sensitivity of diabetic arteries to exogenous norepinephrine was increased under control conditions; maximal contractions were unchanged. Blockade of norepinephrine uptake increased norepinephrine sensitivity more in normal than in diabetic arteries, and there was no longer a significant difference in sensitivity. Thus, under control conditions, neurogenic contractions of the partially denervated diabetic rabbit carotid artery are paradoxically normalized by increased alpha-adrenergic sensitivity of the smooth muscle. The increased sensitivity caused by reduced neuronal uptake can thus preserve neurogenic vasoconstriction and cause supersensitivity to exogenous catecholamines in the sympathetic neuropathy caused by diabetes mellitus.

Influence of the endothelium on tone and the response of isolated pig coronary artery to norepinephrine.

The influence of the endothelium on smooth muscle tone and the response of the pig right coronary artery to norepinephrine (NE) was studied. Isolated rings of artery with and without endothelium were stretched in the presence of nitroprusside to a tension previously determined to be optimal for contraction. During wash out of the nitroprusside, rings without endothelium spontaneously generated tone representing 24% of the contraction caused by potassium (120 mM); in rings with endothelium no significant spontaneous tone was observed. Relaxations were caused by NE in rings with endothelium contracted with prostaglandin F2 alpha (PGF2 alpha). In rings without endothelium, NE relaxed spontaneously generated tone as well as that produced by PGF2 alpha. Independent of the mode or degree of contraction, rings with endothelium were more sensitive to NE than rings without endothelium. The difference in sensitivity to NE between rings with and without endothelium was likely due to endothelial cell alpha-2 adrenoceptors, inasmuch as the difference was abolished by rauwolscine. In the presence of propranolol and prazosin, endothelium-dependent relaxations were observed which were also inhibited by rauwolscine. Nevertheless, beta adrenoceptors are the predominant mediator of the relaxation to NE of pig coronary smooth muscle, because propranolol caused a greater shift to the right of the relaxation induced by NE compared to that caused by endothelium removal. Accordingly, under resting conditions, NE caused contractions only in the presence of propranolol. These contractions were attenuated by prazosin or rauwolscine, but blocked only by a combination of both alpha adrenoceptor antagonists.(ABSTRACT TRUNCATED AT 250 WORDS)

4-Aminopyridine causes apoptosis and blocks an outward rectifier K+ channel in malignant astrocytoma cell lines.

Among the ion channels and pumps activated by growth factor stimulation, K+ channels have been implicated in the growth and proliferation of several cancer cell lines. The role of these channels in central nervous system tumors, however, has not been described. This study used the malignant astrocytoma cell lines U87 and A172. 4-Aminopyridine (4-AP) inhibition of proliferation was dose dependent, and assessment using a TUNEL in situ assay revealed that apoptosis occurred in U87 cells with wild-type p53 but not in A172 cells with mutant p53 (24-hr incubation with mM 4-AP). In patch clamp experiments, we identified two types of K+ currents in both cell lines, a charybdotoxin-sensitive Ca2(+)-activated K+ channel and a 4-AP-sensitive outward rectifier K+ current. The outward rectifier current was blocked by 4-AP in a dose-dependent manner, with half-maximal block occurring at 3.9 mM. The blocking effect of 4 mM 4-AP was noticeable at potentials as low as -65 mV and was statistically significant at -60 mV and above, suggesting that 4-AP-sensitive current is active at physiological potentials. By contrast, charybdotoxin (1 microM) and tetraethylammonium. Cl (2 mM) blocked the Ca2(+)-activated K+ channel in both cell lines but had no appreciable effect on cell growth. Our findings reveal that 4-AP inhibits proliferation and the outward rectifier K+ channel in both U87 and A172 cells. More studies are needed, however, to describe the mechanism by which K+ channels influence proliferation and induce apoptosis.