1 The purpose of this investigation was to determine whether enhanced contractile responses to noradrenaline (NA) of mesenteric arteries from rats with chronic streptozotocin-induced diabetes are associated with increases in mean cytosolic [Ca 2+ ] i . 2 [Ca 2+ ] i was measured with fura 2-AM, and was monitored simultaneously with tension in perfused endothelium-denuded mesenteric arterial rings from 12 ± 14 week diabetic rats and age-and gender-matched control rats. 3 Basal [Ca 2+ ] i (expressed as R n , the normalized fura 2 ratio) was not signi®cantly di erent in arteries from control and diabetic rats. Similarly, no di erences between control and diabetic arteries in the tension or [Ca 2+ ] i responses to 80 mM KCl in the presence of phentolamine were detected. 4 The rate of tension development, peak tension and integrated tension in response to 30 mM NA were all signi®cantly greater in diabetic than control arteries. However, this was not associated with enhancement of the corresponding [Ca 2+ ] i responses in the diabetic arteries. 5 Peak contractile responses to perfusion with both 0.3 and 3 mM NA, but peak [Ca 2+ ] i only in response to 0.3 mM NA, were signi®cantly greater in diabetic than control arteries. 6 NA (30 mM) produced a greater increase in both peak tension and [Ca 2+ ] i in diabetic than control arteries perfused with Ca 2+ -free solution containing 1 mM EGTA. Neither the rate nor the magnitude of NA-induced Ca 2+ in¯ux appeared to be altered in the diabetic arteries. 7 The enhanced sustained contractile response of diabetic arteries to NA appears to be dissociated from increases in [Ca 2+ ] i , and may be due to other factors, such as an increase in the Ca 2+ sensitivity of the contractile proteins.

1 Previous studies from this laboratory have demonstrated that cxl-adrenoceptor-mediated increases in tension and phosphoinositide metabolism are enhanced in the aorta and mesenteric arteries from diabetic rats. The purpose of the present investigation was to determine whether contractile responses to sodium fluoride (NaF), which directly stimulates GTP-binding proteins (G-proteins), are also enhanced in diabetic arteries. 2 NaF (1-20 mM) in the presence of 10 ,UM aluminium chloride produced slowly developing, concentration-dependent contractions in mesenteric arteries from three month streptozotocin-diabetic (60 mg kg-', i.v. ) male Wistar rats and age-matched control rats. The maximum contractile response but not the sensitivity to NaF was significantly greater in mesenteric arteries from diabetic than from control rats, as was the response to noradrenaline (NA). Maximum contractile responses of aorta and caudal artery from diabetic rats to NaF were also significantly enhanced. 3 Removal of the endothelium and denervation with 6-hydroxydopamine did not significantly alter the maximum contractile response of mesenteric arteries from either control or diabetic rats to NaF. Similarly, NaF had no effect on cyclic AMP levels in aorta, and no difference in cyclic AMP levels, either basally or in the presence of NaF, was detected between control and diabetic rat aorta. 4 Contractile responses of mesenteric arteries from both control and diabetic rats to NaF were diminished in calcium-free Krebs solution, but the NaF response remained significantly elevated in mesenteric arteries from diabetic rats compared to control.5 Ryanodine (30 gM) which depletes intracellular calcium stores, nifedipine (3 gM) which blocks dihydropyridine-sensitive calcium channels and calphostin C (0.5 gM) which selectively inhibits protein kinase C, all significantly inhibited maximum contractile responses of mesenteric arteries from control and diabetic rats to NaF. There were no significant differences between control and diabetic arteries in the relative magnitude of the inhibition produced by the three antagonists. 6 These data suggest that there may be increased activation of the same signalling processes that mediate NA-stimulated vasoconstriction, perhaps contraction-associated G-proteins or the effectors coupled to these G-proteins, in response to NaF in mesenteric arteries from diabetic rats. This may also be responsible for the enhanced contractile responses of these arteries to a,-adrenoceptor stimulation.

The mechanisms contributing to the contractile responses to two different concentrations of noradrenaline (NA) in rat aorta and mesenteric artery were compared using nifedipine, which inhibits calcium influx through dihydropyridine-sensitive channels, ryanodine, which depletes intracellular calcium stores, and calphostin C, which inhibits protein kinase C (PKC). Both submaximal and maximal concentrations of NA induced a biphasic response in aorta and mesenteric artery, with an early fast phase and a later sustained tonic component. Calcium release from intracellular stores contributed to the phasic component of contraction to both concentrations of NA in aorta, although to a greater extent to the submaximal concentration. In aorta, inhibiting both intracellular calcium release and calcium influx through dihydropyridine-sensitive channels simultaneously or inhibiting PKC activity alone significantly reduced the tonic response to a maximal concentration of NA. However, the tonic response to a submaximal concentration of NA in aorta was significantly inhibited only when intracellular calcium stores were depleted with ryanodine. In mesenteric artery, the phasic response to a maximal concentration of NA was significantly depressed only when both calcium influx and intracellular calcium release were inhibited simultaneously. However, the phasic response to a submaximal concentration of NA was significantly inhibited by blocking calcium influx alone, but not by blocking intracellular calcium release alone. The tonic component of the contractile response to both concentrations of NA in mesenteric artery appeared to be mediated in part by calcium from both intracellular and extracellular sources.(ABSTRACT TRUNCATED AT 250 WORDS)

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