Protein kinase C-mediated contractile responses of arteries from diabetic rats.

1. The role of protein kinase C (PKC) in mediating enhanced contractile responses of aortae and mesenteric arteries from male rats with 12-14 week streptozotocin-induced diabetes to noradrenaline (NA) was investigated using the PKC activator, phorbol 12,13-dibutyrate (PDB), and the PKC inhibitor, staurosporine. 2. Maximum contractile responses of aortae and mesenteric arteries from diabetic rats to NA were significantly enhanced compared with responses of arteries from age-matched control animals. The maximum NA responses were increased by 59.6 +/- 7.9% in aortae and by 54.9 +/- 7.4% in mesenteric arteries from diabetic animals, compared to their respective controls. 3. Pretreatment of aortae and mesenteric arteries from both control and diabetic animals with staurosporine (5 x 10(-8) M) caused marked inhibition of contractile responses to a maximum concentration of NA (10(-5) M in aortae; 3 x 10(-5) M in mesenteric arteries). In the presence of staurosporine, no difference was observed in the magnitude of contractile responses of arteries from control and diabetic rats to NA. 4. Maximum contractile responses of mesenteric arteries from diabetic rats to PDB were significantly increased (by 45.0 +/- 4.9%) compared to responses of arteries from control animals. In contrast, no significant difference was found in the magnitude of contractile responses or aortae from control and diabetic rats to PDB. 5. Staurosporine (5 x 10(-8) M caused marked attenuation of contractile responses of arteries from control and diabetic rats to a maximum concentration of PDB (3 x 10(-6) M).(ABSTRACT TRUNCATED AT 250 WORDS)     

Contractile responses and calcium mobilization induced by muscarinic agonists in the rat urinary bladder: Effects of age

In the current study, the contractile responses to muscarinic stimulation of urinary bladder strips from young rats and aged rats were compared.

• 1.1. The EC₅₀ values of the two groups in response to bethanechol were similar.
• 2.2. The magnitude and the velocity of tension generation was significantly lower in strips from the aged group.
• 3.3. The magnitude as well as the velocity of tension generation in response to high K⁺ solution of the two models were similar.
• 4.4. The time to peak [Ca²⁺]i in response to bethanechol was prolonged in strips from the aged group as compared with the young group.
• 5.5. ⁴⁵Ca²⁺ influx in response to bethanechol was significantly reduced in the aged group as compared with the young group.

It is concluded that the reduced contractile response to muscarinic stimulation of isolated urinary bladder strips from aged rats is mediated at least in part by a decreased rate of Ca²⁺ entry.     

Enhanced contractile responses of arteries from streptozotocin diabetic rats to sodium fluoride.

1. Previous studies from this laboratory have demonstrated that alpha 1-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 microM aluminium chloride produced slowly developing, concentration-dependent contractions in mesenteric arteries from three month streptozotocin-diabetic (60 mg kg-1, 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 microM) which depletes intracellular calcium stores, nifedipine (3 microM) which blocks dihydropyridine-sensitive calcium channels and calphostin C (0.5 microM) 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 produce by the three antagonist. 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 alpha 1-adrenoceptor stimulation.     

Contractile effects of tacrolimus in human and rat isolated renal arteries

1. We tested the vasoactive properties of the immunosuppressive drug FK 506 (tacrolimus) in preconstricted rat and human isolated renal arteries in vitro. 2. In rat renal arteries, tacrolimus (3, 10 microM) showed a direct and dose-dependent contractile effect by maximally 23 microm (10% of the noradrenaline effect), which was only observed in the presence of intact endothelium. Moreover, a lower concentration of tacrolimus (1 microM) potentiated pressor responses to the sympathetic neurotransmitter noradrenaline but not to ATP in this species. ATP- (0.01-10 microM) induced vasodilation was not affected by tacrolimus (1 microM). 3. In contrast, in human interlobar arteries, tacrolimus failed to induce direct vasoconstriction and did not significantly potentiate constrictor responses to noradrenaline. Acetylcholine-(1 microM) induced vasodilation was much smaller in human than in rat renal arteries suggesting the lack of functional endothelium in the human preparation. 4. The findings suggest that tacrolimus releases an endothelium-derived constricting factor in rat renal arteries to increase vascular tone and to potentiate pressor responses to noradrenaline. In human interlobar arteries, this effect of tacrolimus is not observed probably because of the absence of functional endothelium or the necessary mediator mechanism.     

Contractile effect of ghrelin on isolated guinea-pig renal arteries

Ghrelin, a 28-amino acid peptide, known to exist in both acylated and des-acylated varieties, was identified as the first endogenous ligand of growth hormone secretagogue receptor in 1999. Various arteries are known to express ghrelin receptors, but the direct action of ghrelin on blood vessels has been unclear. In the present study we show that ghrelin concentration-dependently potentiates endothelin-1 (ET-1) induced tension development of guinea-pig renal artery, as measured using a wire-type isometric myography of vascular segments. In vascular smooth muscle cells (SMC) ghrelin caused activation of potassium outward currents via phospholipase C (PLC)-->inositol-1,4,5-trisphosphate (IP3) and PLC-->protein kinase C (PKC) signalling cascade, resulting in hyperpolarizaton of the cell membrane. On a tissue level ghrelin by itself had no effect on isometric tone, but augmented ET-1 induced contraction by a mechanism, involving PLC, Rho-kinase and intracellular IP3 -sensitive Ca2+ release, and not nucleotide-sensitive protein kinases or PKC. Together with our previous findings the data in this study suggest that ghrelin exerts its contractile activity on guinea-pig renal artery by facilitation of ET-1 triggered intracellular signalling in SMC, and/or by stimulating the release of a yet unknown contractile mediator from endothelium.     

Influence of the endothelium on contractile responses of arteries from diabetic rats

Contractile responses of aortas and mesenteric arteries from control and 3 month streptozotocin-diabetic rats to alpha-adrenoceptor agonists were compared in the presence and absence of endothelium. In the presence of endothelium, responses of both arteries from diabetic animals to norepinephrine and methoxamine were enhanced compared to control, although no response to clonidine could be detected in arteries from either control or diabetic animals. Following endothelium removal, no significant differences were found between control and diabetic arteries in maximum contractile responses to noradrenaline or methoxamine. However, the sensitivity (pD2) of diabetic aortas to these two agonists was significantly increased, while maximum responses of diabetic aortas and mesenteric arteries to clonidine were much greater than control. In addition, no differences between control and diabetic aortas were detected when cGMP levels were measured in the absence and presence of acetylcholine. These results suggest that enhanced responsiveness of arteries from diabetic animals to alpha-adrenoceptor stimulation is not the result of a decrease in endothelium-derived relaxing factor (EDRF) release in diabetic blood vessels.     

Contractile and electrical properties of sternohyoid muscle in streptozotocin diabetic rats

1. The effects of diabetes on the electrical and contractile function of skeletal muscle are variable, depending on muscle fibre type distribution. The muscles of the upper airway have a characteristic fibre distribution that differs from previously studied muscles, but the effects of diabetes on upper airway muscle function are unknown. Normally, contraction of upper airway muscles, such as the sternohyoids, dilates and/or stabilizes the upper airway, thereby preventing its collapse. Diabetes is associated with obstructive sleep apnoea in which there is collapse of the upper airway due to failure of the upper airway musculature to maintain airway patency. Therefore, the purpose of the present study was to determine the effects of diabetes on the electrical and contractile characteristics of upper airway muscle. 2. Rats were treated with vehicle (sodium citrate buffer; pH 4.5) or with streptozotocin to induce diabetes, confirmed by the presence of hyperglycaemia, and the contractile and electrical properties of the sternohyoid were compared in these two groups. Isometric contractile properties and membrane potentials were determined in isolated sternohyoid muscles in physiological saline solution at 25 degrees C. 3. Streptozotocin had no effect on sternohyoid muscle fatigue, the tension-frequency relationship or membrane potentials, but did increase contraction time, half-relaxation time, twitch tension and tetanic tension. 4. Streptozotocin-induced diabetes has no effect on sternohyoid muscle fatigue or the tension-frequency relationship, but does reduce contractile kinetics and increases force generation. These effects are not due to changes in resting membrane potential. These data are evidence that the association of sleep apnoea and diabetes is not due to effects on upper airway muscle contractile properties.     

Different mobilization of calcium in endothelin-1-induced contractions in human arteries and veins: effects of calcium antagonists.

We studied the role of extra- and intracellular Ca2+ in endothelin-1-induced contractions of the isolated human internal mammary artery and vein. Veins were more sensitive to the peptide than arteries (concentration shift:3.2-fold; n = 4-10, p less than 0.05). The Ca2+ antagonists darodipine, verapamil, and diltiazem (10(-7)-10(-6) M) did not prevent the response to endothelin-1 in both vessels. In contrast, darodipine (10(-8)-10(-6) M), added after the contraction had developed, partially reversed the response in the artery (26 +/- 7%) and particularly in the vein (67 +/- 5%; n = 4, p less than 0.005 compared to the artery). Removal of extracellular Ca2+ reduced the contractions to endothelin-1 (10(-8) M) in the artery (control: 89 +/- 4% of 100 mM KCl; Ca2(+)-free: 68 +/- 4% n = 4-6, p less than 0.01), but not in the vein except at low concentrations (10(-9) M) of the peptide. After removal of intracellular Ca2+ with caffeine in the artery, endothelin-1 still evoked a contraction (17 +/- 3%, n = 3; p less than 0.005 vs. control), while in the vein the response was abolished. Thus, mobilization of Ca2+ during endothelin-1-induced contractions differs in the human internal mammary artery and vein. In the artery, the contraction depends on extracellular Ca2+, intracellular caffeine-sensitive Ca2+ stores, and a caffeine-insensitive component, while in veins, mobilization of intracellular Ca2+ is most important. Ca2+ antagonists do not prevent, but partially reverse, endothelin-1-induced contractions indicating that voltage-operated Ca2+ channels do not initiate but contribute to the maintenance of the response.     

Time-dependent hyperreactivity to phenylephrine in aorta from untreated diabetic rats: role of prostanoids and calcium mobilization

Diabetes alters vascular smooth muscle contractility. Changes in reactivity to phenylephrine (Phe) in aortas from controls and untreated 1- and 4-week streptozotocin (STZ)-induced diabetic rats were investigated. In 1-week diabetic (DB1) aortas, the maximum response (E(max)) and sensitivity (pD(2)) to Phe were similar to controls (CT1), but in 4-week diabetic (DB4) aortas, the E(max) for Phe was increased compared to CT4 aortas (E(max), DB4: 125+/-8.4% vs. CT4: 89.8+/-4.5%, P<.001). Endothelial denudation increased the response to Phe, and E(max) was increased in the DB4 aortas compared to CT4 (E(max), DB4: 156+/-4.2% vs. CT4: 125+/-3.8%, P<.001). Pretreatment of CT4 and DB4 aortas with indomethacin reduced E(max) and pD(2) for Phe. After indomethacin treatment, no differences in E(max) and pD(2) to Phe were observed in either group. SQ 29548 did not alter the Phe actions in CT4 aortas. However, in DB4 aortas, E(max) was reduced to control level. CT4 and DB4 aortas incubated in free-Ca(2+) solution plus Phe, contracted upon addition of CaCl(2), this response was increased in DB4 aortas. No changes were observed for acetylcholine (ACh) or sodium nitroprusside (SNP) responses. Nitric oxide (NO) release in response to Phe determined by acute L-NAME administration showed no differences in the percentage increase of the contraction in CT1 and DB1 aortas, but was enhanced in DB4 aortas. Results suggested that diabetes induces time-dependent changes in the vascular reactivity to Phe. This response is not related to a reduction of endothelium-derived NO but might be due to an increase in prostaglandin H(2) (PGH(2))/thromboxane A(2) (TxA(2)) and/or an enhanced extracellular Ca(2+) influx.     

Vascular responses of isolated mesenteric resistance and basilar arteries from short- and long-term diabetic rats

Vascular dysfunctions, e.g. alterations in the reactivity of blood vessels to neurotransmitters and hormones, are a well-established complication of diabetes mellitus. Whether these impairments are a consequence of direct postsynaptic deficits and/or indirect presynaptic deficits remains to be determined. To this end, we investigated the influence of the duration of diabetes on relaxation and contraction responses of isolated mesenteric resistance and equally-sized basilar arteries to postsynaptic activation by various vasoactive agents, using streptozotocin-induced diabetic rats and age-matched controls. Relaxation responses to vasodilator agents were studied in KCl-precontracted arteries. The duration of diabetes (4 or 40 weeks) did not affect the vasodilator responses to sodium nitroprusside or salbutamol in either artery. In mesenteric resistance vessels from short-term (4 weeks) and long-term (40 weeks) diabetic rats the vasoconstrictor responses to KCl, serotonin and vasopressin were the same as those in non-diabetic rats; however, the sensitivity (EC₅₀) to noradrenaline was slightly but significantly enhanced after the long-term diabetic state. In contrast to the mesenteric arteries, noradrenaline did not cause contraction in basilar arteries taken from diabetic and control rats. Thus, there appear to be important differences in the reactivity to noradrenaline of the peripheral and cerebral vasculature. The basilar artery from short-term and long-term diabetic rats did not show different responsiveness to vasopressin whereas to serotonin a significant enhanced and decreased sensitivity (EC₁₀ and EC₅₀) was demonstrated in short-term and long-term diabetes, respectively. Our findings indicate that postsynaptic impairments do not play a major role in the alterations of vasoreactivity to vasodilators, noradrenaline or vasopressin seen in experimental diabetes. However, the duration of the diabetic state may have serious consequences for vasoreactivity of basilar arteries to serotonin and, therefore, warrants further investigations. Received: 17 June 1998 / Accepted: 3 August 1998     

糖尿病大鼠病程的不同时期主动脉平滑肌对苯肾上腺素的收缩反应(英文)

目的:研究随着糖尿病的发生发展,大鼠主动脉平滑肌对苯肾上腺素等激动剂收缩反应的变化及其可能机制。方法:用链尿菌素诱导糖尿病后,在第2、6、12周,取主动脉环进行实验观察。结果:苯肾上腺素的浓度依赖性收缩反应曲线,与对照相比:在第2周,低浓度时(0.01-0.03μmol·L~(-1))明显增加(P<0.01),最大反应无明显变化;在第6周,各浓度点均显著增加,且最大收缩反应增加约40％;然而,在第12周1)苯肾上腺素10μmol·L~(-1)引起的最大收缩反应趋向降低(P<0.05),2)在无Ca~(2 )液,也较对照明显减小(P<0.05),3)在无Ca~(2 )液,在尼非地平1μmol· L~(-1)和苯肾上腺素10μmol·L~(-1)存 在下,复Ca~(2 )引起的收缩在两组间的差异未见显著性,4)在正常Krebs’液,环匹阿尼酸10μmol·L~(-1)引起的收缩反应较对照也显著减小(P<0.001)。结论:(1)在糖尿病的第2周,平滑肌α_1-肾上腺素能受体的敏感性增加。(2)糖尿病大鼠主动脉平滑肌对苯肾上腺素收缩反应的异常变化,与通过电压依赖性钙通道的Ca~(2 )内流大小、胞内功能性Ca~(2 )池大小及其胞内Ca~(2 )池耗竭后所引起的充电性内流变化密切相关。     

Endothelium-dependent relaxation in isolated renal arteries of diabetic rabbits

1 In this study, we have investigated the vasodilator response to acetylcholine under diabetes conditions in isolated renal arteries of rabbits. We have also examined the contribution of endothelium-derived nitric oxide (EDNO) and endothelium-derived hyperpolarizing factor (EDHF) to the endothelium-dependent relaxation caused by acetylcholine in the renal arteries of alloxan-induced diabetic rabbits. 2 Acetylcholine (10(-10) - 10(-4) M) produced cumulative concentration-response curve in the renal arteries of both control and diabetic rabbits. The EC50 values and maximal responses to acetylcholine were not significantly different relative to diabetic conditions. In order to isolate the EDHF component of acetylcholine-induced vasodilator response, L-nitro-methyl arginine ester (L-NAME, 10(-4) M) and indomethacin (10(-6) M) were added to the Krebs' solution throughout the experiment. Under these conditions, acetylcholine induced vasodilatation in the isolated renal arteries from both control and diabetic rabbits. The vasodilator response to acetylcholine was not affected under diabetic conditions. 3 Sodium nitroprusside (SNP)-induced relaxation was increased in the diabetic rabbits compared with the control animals. 4 Tetrabutyl ammonium (TBA, 0.5 mM) produced a significant reduction in acetylcholine-induced vasodilatation in both preparations from control and diabetic animals, consistent with involvement of K+ channels in mediating this response. Glibenclamide (1 microM) attenuated acetylcholine-induced vasodilatation in preparations from control animals only, while iberiotoxin (0.05 microM) significantly reduced the vasodilator response to acetylcholine in preparations from both control and diabetic animals. 5 The role of EDNO in mediating acetylcholine-induced vasodilatation was examined. The vascular preparations were incubated with 20 mM K(+)-Krebs' solution to inhibit the EDHF contribution to acetylcholine-induced vasodilatation. Under this condition, acetylcholine induced a vasodilator response in both preparations from control and diabetic rats. Pretreatment with L-NAME (10(-4) M) attenuated acetylcholine-induced vasodilatation in both preparations, indicating an nitric oxide-mediated vasodilator response. 6 Our results indicated that acetylcholine-induced vasodilatation in the isolated renal arteries of alloxan-induced diabetic rabbits was not affected under diabetic conditions. Acetylcholine-induced vasodilatation is mediated by two vasodilator components; namely, EDHF and EDNO. The contribution of EDHF and EDNO to acetylcholine-induced vasodilatation was not affected under diabetic conditions and there was no indication of endothelial dysfunction associated with diabetes. EDHF component was found to act mainly through high conductance Ca(2+)-activated K+ channels under normal and diabetic conditions, while the adenosine triphosphate-dependent K+ channels were involved in mediating acetylcholine vasodilator response in the control preparations only.     

Augmented inositol phosphate production in mesenteric arteries from diabetic rats.

The effects of noradrenaline (NA) on contraction and phosphoinositide metabolism were compared in mesenteric arteries from rats with chronic streptozotocin-induced diabetes and from age-matched control rats. Maximum contractile responses of mesenteric arteries from diabetic rats to NA (30 microM) were significantly greater than control in both the presence and absence of extracellular Ca2+. Basal incorporation of [3H]myoinositol into total [3H]inositol phosphates was greater in diabetic than control mesenteric arteries. NA (30 microM) resulted in a time-dependent increase in total [3H]inositol phosphate production, which was also significantly greater in diabetic than in control preparations. The increase in total [3H]inositol phosphates produced by NA in both control and diabetic arteries was blocked by the alpha 1-adrenoceptor antagonist, prazosin. Absolute levels of inositol 1,4,5-trisphosphate (I(1,4,5)P3), measured by protein binding assay, were also increased in response to 30 microM NA in both control and diabetic arteries. Although basal I(1,4,5)P3 levels were not significantly different, NA-induced increases in I(1,4,5)P3 were significantly greater in diabetic than in control arteries at each time-point measured. These data indicate that phosphoinositide metabolism is enhanced in mesenteric arteries from rats with chronic streptozotocin-induced diabetes in response to a maximum concentration of NA. Augmented production of the second messengers I(1,4,5)P3 and, presumably, 1,2-diacylglycerol may contribute to the enhanced maximum contractile responses of the diabetic arteries to NA.     

Altered responses to calcium and trifluoperazine in K+-depolarized duodenum from alloxan diabetic rats

Contractile responses to calcium were examined in the K+-depolarized duodenum from normal and alloxan-diabetic rats. In addition, verapamil and trifluoperazine which are well-known calcium channel blocker and calmodulin inhibitor respectively were used as tools in order to approach to the mechanism of changes resulting from diabetes. Decreased contractile responses to calcium were observed in the alloxan diabetic rat duodenum compared to normals. Trifluoperazine-induced non-competitive inhibition was significantly affected depending on diabetes, while verapamil-induced competitive inhibition was not changed. The non-competitive inhibition affinity constant for trifluoperazine was significantly elevated in the K+-depolarized duodenum from alloxan-diabetic rats. On the basis of findings obtained in this study, possible mechanism of the effect of experimental diabetes on the calcium-induced contraction is discussed.     

Contractile response of human omental arteries to endothelin.

The effects of endothelin have been studied in isolated arterial segments (0.8-1 mm in external diam.) of human omental arteries obtained during the course of abdominal operations (15 patients, 7 men and 8 women). Paired segments, one normal and the other de-endothelized, were mounted for isometric recording of tension in organ baths. Endothelin produced concentration-dependent contractions with an EC50 value of 5.4 x 10(-9) M. Removal of endothelium did not affect significantly endothelin-induced contractions (EC50, 6.7 x 10(-9) M). Removal of extracellular calcium or addition of the calcium channel blocker nicardipine (10(-6) M) diminished but did not abolish responses to endothelin. These results indicate that endothelin exerts powerful contractile effects on human isolated omental arteries which are independent of the presence of an intact endothelial cell layer; this contraction cannot be explained solely by voltage-dependent calcium channels.     

Impaired endothelium-dependent relaxation in isolated resistance arteries of spontaneously diabetic rats.

1. Previous studies have shown that endothelium-dependent relaxation in the aorta of spontaneously diabetic bio bred rats (BB) is impaired. 2. We have investigated noradrenaline (NA) contractility, endothelium-dependent acetylcholine (ACh) and bradykinin (BK) relaxation, and endothelium-independent sodium nitroprusside (SNP) relaxation in mesenteric resistance arteries of recent onset BB rats and established insulin treated BB rats, compared to their age-matched non diabetic controls. 3. There was no significant difference in the maximum contractile response or sensitivity to noradrenaline in either of the diabetic groups compared to their age-matched controls. 4. Incubation with the nitric oxide synthetase inhibitor NG-nitro-L-arginine (L-NOARG) resulted in a significant increase in maximum contractile response to noradrenaline in the recent onset age-matched control group (P < 0.05). Analysis of the whole dose-response curve (using ANOVA for repeated measures with paired t test) showed a significant left-ward shift following the addition of L-NOARG (P < 0.001). A similar but less marked shift (P < 0.01) was evident in vessels from recent onset diabetics. An overall shift in both sensitivity and maximum response was also evident in the age-matched non diabetic controls of the insulin-treated group (P < 0.05). However, by contrast, there was no significant change in sensitivity in the insulin-treated diabetic rats. 5. ACh-induced endothelium-dependent relaxation was significantly impaired in the recent onset diabetic rats compared to their age-matched controls (47 +/- 11% versus 92 +/- 2%, P < 0.05, n = 6), and in the insulin treated diabetic rats (34 +/- 5% versus 75 +/- 6%, P < 0.05, n = 6). The relaxation responses to BK also were significantly impaired in the diabetic rats compared to their age-matched controls (recent onset: 20 +/- 3% versus 72 +/- 7%, P < 0.05, n = 6; insulin treated: 12 +/- 9% versus 68 +/- 7%, P < 0.05, n = 7). 6. Incubation with either the nitric oxide synthetase substrate, U-arginine, or the free radical scavenging enzyme superoxide dismutase (150 mu ml-1) failed to improve the attenuated response of acetylcholine-induced relaxation in the diabetic vessels. 7. Endothelium-dependent relaxation mediated by ACh and BK was significantly attenuated in both the diabetic and control vessels after incubation with L-NOARG. 8. Pretreatment with a cyclo-oxygenase inhibitor, indomethacin, significantly enhanced the relaxation to ACh in both the recent onset and insulin treated diabetic rats (42 +/- 10%, n = 7 versus 64 +/- 7%, n = 7, P < 0.05, and 40 +/- 5%, n = 7 versus 65 +/- 9%, n = 6, P < 0.05). 9. Following endothelium removal, there was a marked impairment in endothelium-dependent relaxation responses to ACh and BK in both the diabetic and control vessels. 10. Incubation with the thromboxane A2 receptor antagonist SQ29548, did not significantly improve the ACh endothelium-dependent relaxation response in the diabetic vessels. 11. Endothelium-independent relaxation to sodium nitroprusside was significantly impaired in the first group of diabetic vessels studied; however, subsequent studies showed no impairment of the sodium nitroprusside response in the diabetic vessels. 12. In conclusion, the ability of the endothelium to regulate vascular contractility is reduced in recent onset diabetic vessels, and significantly impaired in established insulin treated diabetics. Relaxation to the endothelium-dependent vasodilators ACh and BK was impaired in both the recent onset and the established insulin treated diabetics, and the ACh response was significantly improved following pretreatment with indomethacin, suggesting a role for a cyclo-oxygenase-derived vasoconstrictor. Preliminary studies with a thromboxane A2, receptor antagonist, SQ29548 did not significantly improve the impaired relaxation to ACh, indicating that the vasoconstrictor prostanoid is not thromboxane A2.     

Calcium accumulation in liver and calcium mobilization in bone of lead-poisoned rats

Administration of lead decreased ⁴⁷Ca radioactivity in bone, increased the radioactivity in liver and did not alter the radioactivity in blood. Fecal excretion of ⁴⁷Ca was increased and urinary excretion was lower after administration of lead. The results demonstrate that lead mobilizes calcium in the bone.     

银杏叶提取物对糖尿病大鼠肾脏细胞色素C、线粒体Ca2+的影响

目的:探讨银杏叶提取物对Ⅱ型糖尿病大鼠肾脏病变期间肾脏线粒体钙、细胞色素C水平的影响.方法:将成龄雄性大鼠腹腔注射链脲佐菌素制成Ⅱ型糖尿病模型,30只大鼠随机分为对照组、糖尿病模型组和银杏叶提取物治疗组,用张均田的改良方法测定细胞色素C含量;用火焰原子吸收法检测线粒体钙含量.结果:银杏叶提取物治疗组动物的细胞色素C、线粒体钙与模型组相比差异显著(P＜0.05).结论:银杏叶提取物治疗减少线粒体释放细胞色素C,维持线粒体钙稳态.     

Rho kinase and protein kinase C involvement in vascular smooth muscle myofilament calcium sensitization in arteries from diabetic rats

Background and purpose:

Diabetes mellitus (DM) causes multiple dysfunctions including circulatory disorders such as cardiomyopathy, angiopathy, atherosclerosis and arterial hypertension. Rho kinase (ROCK) and protein kinase C (PKC) regulate vascular smooth muscle (VSM) Ca²⁺ sensitivity, thus enhancing VSM contraction, and up-regulation of both enzymes in DM is well known. We postulated that in DM, Ca²⁺ sensitization occurs in diabetic arteries due to increased ROCK and/or PKC activity.

Experimental approach:

Rats were rendered hyperglycaemic by i.p. injection of streptozotocin. Age-matched control tissues were used for comparison. Contractile responses to phenylephrine (Phe) and different Ca²⁺ concentrations were recorded, respectively, from intact and chemically permeabilized vascular rings from aorta, tail and mesenteric arteries.

Key results:

Diabetic tail and mesenteric arteries demonstrated markedly enhanced sensitivity to Phe while these changes were not observed in aorta. The ROCK inhibitor HA1077, but not the PKC inhibitor chelerythrine, caused significant reduction in sensitivity to agonist in diabetic vessels. Similar changes were observed for myofilament Ca²⁺ sensitivity, which was again enhanced in DM in tail and mesenteric arteries, but not in aorta, and could be reduced by both the ROCK and PKC blockers.

Conclusions and implications:

We conclude that in DM enhanced myofilament Ca²⁺ sensitivity is mainly manifested in muscular-type blood vessels and thus likely to contribute to the development of hypertension. Both PKC and, in particular, ROCK are involved in this phenomenon. This highlights their potential usefulness as drug targets in the pharmacological management of DM-associated vascular dysfunction.     

Contractile responses to calcium chloride in rat aortic rings bathed in K+-free solution are resistant to organic calcium antagonists.

1. In rat aortic rings, devoid of functional endothelium, suspended in a modified Krebs solution (KCl: 0 mM; CaCl2: 0.63 mM), addition of CaCl2 (0.89-10 mM) produced concentration-related increases in tension (Emax = 2.38 +/- 0.10 g, EC50 = 2.31 +/- 0.15 mM, n = 36). 2. The Ca2+ evoked contractile responses were not modified by cinnarizine (10 microM), diltiazem (1 microM), ryanodine (10 microM), verapamil (1 microM), or the dihydropyridines, nitrendipine (1 microM) and (-)-Bay K 8644 (0.003-0.3 microM). 3. Cobalt chloride (0.1-1 mM) competitively antagonized the Ca2+ concentration-response curve; the Schild plot (slope 1.08 +/- 0.04), gave a pA2 value of 3.3 +/- 0.01 (n = 27). Nickel chloride (0.5-1 mM) displaced the Ca2+ concentration-response curve to the right, without an effect on the maximum response. Cadmium chloride (3-30 microM) depressed the maxima of concentration-response curves to Ca2+ with an IC50 of 15.5 +/- 1.1 microM (n = 6). 4. Monochlorobenzamil (100 microM), a Na+-Ca2+ exchange inhibitor, failed to modify the Ca2+-induced contractions. 5. In conclusion, Ca2+ evoked concentration-related contractile responses of rat aortic rings bathed in a K+-free medium; these effects were attenuated by the divalent cations cobalt, nickel and cadmium, but not modified by several organic calcium antagonists. The lack of effect of diltiazem verapamil and the dihydropyridines would suggest that, under these experimental conditions, extracellular Ca2+ enters the cytosol via pathways which are distinct from the slow (L-type) calcium channels.