Prasad V.G. Katakam

Endothelial dysfunction precedes hypertension in diet-induced insulin resistance

The insulin-resistant (IR) syndrome may be an impetus for the development of hypertension (HTN). Unfortunately, the mechanism by which this could occur is unclear. Our laboratory and others have described impaired endothelium-mediated relaxation in IR, mildly hypertensive rats. The purpose of the current study is to determine if HTN is most likely a cause or result of impaired endothelial function. Sprague-Dawley rats were randomized to receive a fructose-rich diet for 3, 7, 10, 14, 18, or 28 days or were placed in a control group. The control group received rat chow. After diet treatment, animals were instrumented with arterial cannulas, and while awake and unrestrained, their blood pressure (BP) was measured. Subsequently, endothelium-mediated relaxation to acetylcholine was determined (in vitro) by measuring intraluminal diameter of phenylephrine-preconstricted mesenteric arteries ( approximately 250 microM). Serum insulin levels were significantly elevated in all groups receiving fructose feeding compared with control, whereas there were no differences in serum glucose levels between groups. Impairment of endothelium-mediated relaxation starts by day 14 [mean percent maximal relaxation (Emax): 69 +/- 10% of baseline] and becomes significant by day 18 (Emax: 52 +/- 11% of baseline; P < 0.01). However, the mean BP (mmHg) does not become significantly elevated until day 28 [BP: 132 +/- 1 (day 28) vs. 116 +/- 3 (control); P < 0.05]. These findings demonstrate that both IR and endothelial dysfunction occur before HTN in this model and suggest that endothelial dysfunction may be a mechanism linking insulin resistance and essential HTN.The insulin-resistant (IR) syndrome may be an impetus for the development of hypertension (HTN). Unfortunately, the mechanism by which this could occur is unclear. Our laboratory and others have described impaired endothelium-mediated relaxation in IR, mildly hypertensive rats. The purpose of the current study is to determine if HTN is most likely a cause or result of impaired endothelial function. Sprague-Dawley rats were randomized to receive a fructose-rich diet for 3, 7, 10, 14, 18, or 28 days or were placed in a control group. The control group received rat chow. After diet treatment, animals were instrumented with arterial cannulas, and while awake and unrestrained, their blood pressure (BP) was measured. Subsequently, endothelium-mediated relaxation to acetylcholine was determined (in vitro) by measuring intraluminal diameter of phenylephrine-preconstricted mesenteric arteries (∼250 μM). Serum insulin levels were significantly elevated in all groups receiving fructose feeding compared with control, whereas there were no differences in serum glucose levels between groups. Impairment of endothelium-mediated relaxation starts by day 14 [mean percent maximal relaxation (Emax): 69 ± 10% of baseline] and becomes significant by day 18 (Emax: 52 ± 11% of baseline; P < 0.01). However, the mean BP (mmHg) does not become significantly elevated until day 28 [BP: 132 ± 1 ( day 28) vs. 116 ± 3 (control); P < 0.05]. These findings demonstrate that both IR and endothelial dysfunction occur before HTN in this model and suggest that endothelial dysfunction may be a mechanism linking insulin resistance and essential HTN.

Dihydroxyeicosatrienoic acids are potent activators of Ca2+‐activated K+ channels in isolated rat coronary arterial myocytes

1 Dihydroxyeicosatrienoic acids (DHETs), which are metabolites of arachidonic acid (AA) and epoxyeicosatrienoic acids (EETs), have been identified as highly potent endogenous vasodilators, but the mechanisms by which DHETs induce relaxation of vascular smooth muscle are unknown. Using inside‐out patch clamp techniques, we examined the effects of DHETs on the large conductance Ca2+‐activated K+ (BK) channels in smooth muscle cells from rat small coronary arteries (150–300 μm diameter). 2 11,12‐DHET potently activated BK channels with an EC50 of 1.87 ± 0.57 nm (n= 5). Moreover, the three other regioisomers 5,6‐, 8,9‐ and 14,15‐DHET were equipotent with 11,12‐DHET in activating BK channels. The efficacy of 11,12‐DHET in opening BK channels was much greater than that of its immediate precursor 11,12‐EET. In contrast, AA did not significantly affect BK channel activity. 3 The voltage dependence of BK channels was dramatically modulated by 11,12‐DHET. With physiological concentrations of cytoplasmic Ca2+ (200 nm), the voltage at which the channel open probability was half‐maximal (V1/2) was shifted from a baseline of 115.6 ± 6.5 mV to 95.0 ± 10.1 mV with 5 nm 11,12‐DHET, and to 60.0 ± 8.4 mV with 50 nm 11,12‐DHET. 4 11,12‐DHET also enhanced the sensitivity of BK channels to Ca2+ but did not activate the channels in the absence of Ca2+. 11,12‐DHET (50 nm) reduced the Ca2+ EC50 of BK channels from a baseline of 1.02 ± 0.07 μm to 0.42 ± 0.11 μm. 5 Single channel kinetic analysis indicated that 11,12‐DHET did not alter BK channel conductance but did reduce the first latency of BK channel openings in response to a voltage step. 11,12‐DHET dose‐dependently increased the open dwell times, abbreviated the closed dwell times, and decreased the transition rates from open to closed states. 6 We conclude that DHETs hyperpolarize vascular smooth muscle cells through modulation of the BK channel gating behaviour, and by enhancing the channel sensitivities to Ca2+ and voltage. Hence, like EETs, DHETs may function as endothelium‐derived hyperpolarizing factors.

EDHF-mediated relaxation is impaired in fructose-fed rats.

Insulin resistance (IR) is associated with endothelial dysfunction. A defect in endothelium-dependent relaxation via outward potassium conductance has been observed in mesenteric arteries from IR rats. The purpose of this study was to assess whether this defect in endothelium-dependent relaxation was due to impaired endothelium-derived hyperpolarizing factor (EDHF) and to determine which specific potassium channel(s) are involved in relaxation. This was accomplished by using specific potassium channel inhibitors in the presence of nitric oxide synthase and cyclooxygenase inhibition. In addition, we sought to assess the function of smooth muscle cell adenosine triphosphate (ATP)-dependent potassium (K(ATP)) channels. Sprague-Dawley rats were randomized to control or IR. To determine EDHF-mediated relaxation, acetylcholine (ACh)-induced (10(-9)-10(-5) M) relaxation was measured (in vitro) in mesenteric arteries in the presence of indomethacin (10(-5) M) and N-nitro-L-arginine (L-NNA) (10(-4) M). Subsequently the combination of charybdotoxin (CTX) (0.1 microM) and apamin (0.5 microM) or glibenclamide (Glib) (10 microM) was added to the bath to inhibit KCa or K(ATP), respectively. In separate experiments, relaxation to pinacidil (10(-13)-10(-5) M), a K(ATP) activator, was assessed in vessels with intact endothelium, endothelium denuded, or with L-NNA. Maximal relaxation to ACh in the presence of L-NNA and indomethacin was 68+/-6% for control and 12+/-3% for IR (p<0.01). The addition of CTX + apamin almost abolished EDHF-mediated relaxation in control (Emax, 8+/-5% vs. 68+/-6%; p<0.01), whereas Glib had little affect. Neither CTX + apamin nor Glib had any affect on IR. Additionally, IR arteries were less sensitive to pinacidil than were controls (EC50, 1.5+/-0.9 microM vs. 5x10(-4)+/-3x10(-4) microM, respectively; p<0.01). Endothelial removal or L-NNA pretreatment of control arteries decreased the response to pinacidil similar to IR, whereas IR vessels were unaffected. EDHF-mediated relaxation is impaired in IR arteries. In addition, the K(Ca) channel appears to be imperative for activity of EDHF in rat small mesenteric arteries. Moreover, activation of K(ATP) channels by pinacidil is impaired in IR, and this appears to be a result of endothelial dysfunction.

Cytochrome P450 Activity and Endothelial Dysfunction in Insulin Resistance

Impaired endothelium-dependent relaxation attributable to nitric oxide/prostacyclin-independent factor (endothelium-dependent hyperpolarizing factor; EDHF) has been demonstrated in the small mesenteric arteries of insulin-resistant rats. The purpose of this study was to determine if modulation of the cytochrome P450 enzyme system would restore EDHF-mediated relaxation in insulin-resistant rats. Sprague-Dawley rats were randomized to control (n = 32) or insulin-resistant (n = 32) groups. Each group was further randomized to treatment (n = 48) or placebo (n = 16). Miconazole (3 days) and phenobarbital (3 and 14 days) achieved cytochrome P450 inhibition and induction, respectively. Following drug treatment, mean arterial pressure was measured and vascular function was assessed in small mesenteric arteries in vitro. Specifically, acetylcholine-induced relaxation alone and in the presence of indomethacin plus N-nitro-L-arginine (LNNA) or KCl was determined. Miconazole reduced the maximal relaxation in response to acetylcholine in control rats. Similarly, in the presence of LNNA plus indomethacin, acetylcholine-induced relaxation was impaired in the miconazole-treated control group versus the placebo group, whereas relaxation in the presence of KCl was unchanged. Miconazole did not affect relaxation in insulin-resistant arteries. In contrast, 3- and 14-day treatment with phenobarbital significantly improved acetylcholine-induced relaxation in insulin-resistant arteries. Likewise, acetylcholine-mediated relaxation in the presence of LNNA plus indomethacin was also improved after phenobarbital treatment, while relaxation in the presence of KCl was unchanged. Phenobarbital treatment did not affect the control group. Miconazole treatment increased the mean arterial pressure in control rats, while 14-day phenobarbital treatment normalized the mean arterial pressure in insulin-resistant rats. Cytochrome P450 induction results in the restoration of EDHF-mediated relaxation in small mesenteric arteries and the normalization of mean arterial pressure in insulin-resistant rats. Thus, endothelial dysfunction secondary to insulin resistance can be reversed by the induction of cytochrome P450.

Mitochondrial Mechanisms in Cerebral Vascular Control: Shared Signaling Pathways with Preconditioning

Mitochondrial-initiated events protect the neurovascular unit against lethal stress via a process called preconditioning, which independently promotes changes in cerebrovascular tone through shared signaling pathways. Activation of adenosine triphosphate (ATP)-dependent potassium channels on the inner mitochondrial membrane (mitoKATP channels) is a specific and dependable way to induce protection of neurons, astroglia, and cerebral vascular endothelium. Through the opening of mitoKATP channels, mitochondrial depolarization leads to activation of protein kinases and transient increases in cytosolic calcium (Ca2+) levels that activate terminal mechanisms that protect the neurovascular unit against lethal stress. The release of reactive oxygen species from mitochondria has similar protective effects. Signaling elements of the preconditioning pathways also are involved in the regulation of vascular tone. Activation of mitoKATP channels in cerebral arteries causes vasodilation, with cell-specific contributions from the endothelium, vascular smooth muscles, and nerves. Preexisting chronic conditions, such as insulin resistance and/or diabetes, prevent preconditioning and impair relaxation to mitochondrial-centered responses in cerebral arteries. Surprisingly, mitochondrial activation after anoxic or ischemic stress appears to protect cerebral vascular endothelium and promotes the restoration of blood flow; therefore, mitochondria may represent an important, but underutilized target in attenuating vascular dysfunction and brain injury in stroke patients.

Impaired Mitochondrial Respiration in Large Cerebral Arteries of Rats with Type 2 Diabetes.

Mitochondrial dysfunction has been suggested as a potential underlying cause of pathological conditions associated with type 2 diabetes (T2DM). We have previously shown that mitochondrial respiration and mitochondrial protein levels were similar in the large cerebral arteries of insulin-resistant Zucker obese rats and their lean controls. In this study, we extend our investigations into the mitochondrial dynamics of the cerebral vasculature of 14-week-old Zucker diabetic fatty obese (ZDFO) rats with early T2DM. Body weight and blood glucose levels were significantly higher in the ZDFO group, and basal mitochondrial respiration and proton leak were significantly decreased in the large cerebral arteries of the ZDFO rats compared with the lean controls (ZDFL). The expression of the mitochondrial proteins total manganese superoxide dismutase (MnSOD) and voltage-dependent anion channel (VDAC) were significantly lower in the cerebral microvessels, and acetylated MnSOD levels were significantly reduced in the large arteries of the ZDFO group. Additionally, superoxide production was significantly increased in the microvessels of the ZDFO group. Despite evidence of increased oxidative stress in ZDFO, exogenous SOD was not able to restore mitochondrial respiration in the ZDFO rats. Our results show, for the first time, that mitochondrial respiration and protein levels are compromised during the early stages of T2DM.

Cerebrovascular function and mitochondrial bioenergetics after ischemia-reperfusion in male rats

The underlying factors promoting increased mitochondrial proteins, mtDNA, and dilation to mitochondrial-specific agents in male rats following tMCAO are not fully elucidated. Our goal was to determine the morphological and functional effects of ischemia/reperfusion (I/R) on mitochondria using electron microscopy, Western blot, mitochondrial oxygen consumption rate (OCR), and Ca2+ sparks activity measurements in middle cerebral arteries (MCAs) from male Sprague Dawley rats (Naïve, tMCAO, Sham). We found a greatly increased OCR in ipsilateral MCAs (IPSI) compared with contralateral (CONTRA), Sham, and Naïve MCAs. Consistent with our earlier findings, the expression of Mitofusin-2 and OPA-1 was significantly decreased in IPSI arteries compared with Sham and Naïve. Mitochondrial morphology was disrupted in vascular smooth muscle, but morphology with normal and perhaps greater numbers of mitochondria were observed in IPSI compared with CONTRA MCAs. Consistently, there were significantly fewer baseline Ca2+ events in IPSI MCAs compared with CONTRA, Sham, and Naïve. Mitochondrial depolarization significantly increased Ca2+ sparks activity in the IPSI, Sham, Naïve, but not in the CONTRA group. Our data indicate that altered mitochondrial structure and function occur in MCAs exposed to I/R and that these changes impact not only OCR but Ca2+ sparks activity in both IPSI and CONTRA MCAs.