The polycationic aminoglycosides modulate the vasoconstrictive effects of endothelin: relevance to cerebral vasospasm

Abstract

1. The vasoactive peptide endothelin (ET) has been implicated in the pathogenesis of cerebral vasospasm following subarachnoid haemorrhage. In these studies we investigated the involvement of protein kinase C (PKC) in sustained vasoconstriction induced by ET-1 in canine cerebral arteries. We also examined the ability of the aminoglycoside antibiotics to reverse the effects mediated by ET-1 in canine cerebrovascular smooth muscle cells (CVSMC). 2. The ET(A) receptor antagonist, BQ-123, showed a competitive inhibition of the ET-1 responses. 3. The vasoconstrictor action of both ET-1 (0.5 nM) and phorbol myristate acetate (PMA) (160 nM) was reversed by a selective PKC inhibitor, Ro-32-0432. 4. In cerebral arteries precontracted with ET-1 the aminoglycosides caused a concentration-dependent relaxation. The EC(50s) for the relaxation were as follows: 0.54+/-0.05, 0.63+/-0.01, 1.88+/-0.46 and 2.3+/-0.92 mM for gentamicin, neomycin, streptomycin and kanamycin, respectively. 5. Gentamicin caused a concentration-dependent decrease of the PMA-induced responses in calcium free medium. 6. PKC activity was elevated in CVSMC exposed to ET-1 (170%) and PMA (167%) for a period of time (60 min) corresponding to maximum tonic contraction induced by these agents in arterial rings. 7. The administration of the aminoglycosides to CVSMC, in concentrations corresponding to the EC(50s) from contractility studies, reduced the effects of both ET-1 and PMA on PKC activity to the levels not different from controls. 8. These results show that the aminoglycosides are able to inhibit sustained vasoconstriction induced by ET-1, an effect which is due, at least in part, to the inhibition of PKC.

Figure 1

Concentration-effect curves for the vasoconstriction of rings of basilar artery elicited by endothelin-1 (0.5 nM) in the absence or presence of a selective antagonist of ET_A receptor, BQ-123 (0.1 μM). Values are expressed as a percentage (% control) of the response observed in the absence of the antagonist. Data points represent means±s.e.mean (bars) for measurements with four to five individual ring preparations.

Figure 2

Effects of the PKC inhibitor, Ro-32-0432, on vasoconstriction induced in rings of basilar artery by endothelin-1 (ET-1) (0.5 nM) and PMA (160 nM). The ring preparations were first exposed to ET-1 or PMA, and Ro-32-0432 was administered after tonic response to the agents had developed. The responses are expressed as the percentage of maximum tonic tension observed in the absence of inhibitor (ordinate). Each bar represents mean±s.e.mean of 4 – 5 experiments. ^***P<0.001, compared with control responses.

Figure 3

Representative traces of the effects of gentamicin on the responses of ring preparations pretreated with ET-1 (a) or PMA (b). Arrows indicate time of administration of ET-1, PMA and gentamicin. Each tracing illustrating the response of an individual preparation is representative of three to five independently conducted experiments.

Figure 4

Cumulative concentration-effect curves for the relaxation of rings of canine basilar artery produced by the aminoglycosides. The preparations were first exposed to ET-1 (0.5 nM) (a) or PMA (160 nM) (b) and the aminoglycosides, gentamicin (genta), neomycin (neo), kanamycin (kana) and streptomycin (strepto), were administered in increasing cumulative concentrations to preparations in which maximum tonic tension had developed. Ordinate: relaxation expressed as a percentage of the maximum tension induced by ET-1. Abscissa: concentration of the aminoglycosides on a logarithmic scale. Data points represent the means±s.e.mean for experiments conducted with five or more ring preparations from three or more animals.

Figure 5

Representative traces of the vasoconstrictor responses induced by PMA (160 nM) in calcium free medium. Gentamicin (a) and Ro-32-0432 (b) were administered to the preparations in which maximum tonic contraction to PMA had developed. (c) shows representative traces of the effects of bradykinin on rings of basilar artery with denuded endothelium. The rings were precontracted with 5-HT (10 μM). Bradykinin produced no relaxation suggesting that endothelium had been removed from the preparations. Each trace, which illustrates the response of an individual ring preparation, is representative of three or four independent experiments.

Figure 6

Effects of the aminoglycosides on protein kinase C (PKC) activity in cultured canine cerebrovascular smooth muscle cells. PKC activity was determined in the soluble and particulate fractions of serum starved and (48 h) cells exposed to ET-1 (0.5 nM) (a) or PMA (160 nM) (b) for 60 min. The aminoglycosides, gentamicin (genta), neomycin (neo), kanamycin (kana) and streptomycin (strepto), were administered 30 min after ET-1 application, at the concentrations corresponding to their EC_50s determined in contractility experiments. Each bar represents mean value±s.e.mean of six experiments. ^**P<0.01 vs control; #P<0.01 vs stimulation with ET-1 alone; ##P<0.001 vs stimulation with ET-1 alone.