1. Although multiple cytochrome P450s (CYP) contribute to hepatic phase I metabolism, CYP3A is the principal subfamily present in human and mouse small intestine. 2. Differences in phase I metabolism were investigated using midazolam (MDZ) hydroxylation in mouse liver and intestinal microsomes. The net MDZ metabolite formation rate in intestinal microsomes was approximately 30% that of liver microsomes (at 250 micro M MDZ). 3. Quantitative Western blotting with anti-CYP3A1 antibody detected two bands of immunoreactive protein in both liver and intestinal samples, 2.24 +/- 0.27 (mean +/- SD, n = 3) and 0.64 +/- 0.08 pmol mg(-1) protein, respectively. Qualitative Western blotting with anti-CYP2C11 antibody detected a single band of immunoreactive protein in liver microsomes and no signal in intestinal samples (1 micro g sample). 4. Ketoconazole potently inhibited formation of both alpha- and 4-OH-MDZ metabolites in intestinal microsomes (IC(50)' of 0.126 +/- 0.010 and 0.0955 +/- 0.014 micro M, respectively) and of 4-OH-MDZ formation in mouse liver microsomes (IC(50) of 0.041 +/- 0.003 micro M). However, ketoconazole (5 micro M) did not produce 50% inhibition of alpha-OH-MDZ formation in mouse liver microsomes. Inhibition by ritonavir (5 micro M) produced similar results. 5. MDZ hydroxylation is predominately CYP3A dependent in mouse intestine (compared with mouse liver) since CYP2C is not expressed in the intestine. The importance of CYP3A in the mouse intestine appears to mirror that in humans.
Cytosolic Ca2+ and protein kinase C (PKC) may regulate the myogenic contraction of arterial myocytes. The role of these second messengers is examined in skeletal muscle small arteries, which have strong myogenic activity, and mesenteric small arteries, which have weak myogenic activity. The vessels were isolated and cannulated. The inner diameter was measured with a video-digitizing system. Cytosolic Ca2+ concentration was assessed by fura 2. Skeletal muscle small arteries dilated from 122 +/- 6 to 153 +/- 6 microm immediately after the transmural pressure change from 40 to 100 mmHg and constricted to 121 +/- 5 microm (myogenic contraction) with an increase in the 340/380 fluorescence ratio (by approximately 33%) in control vessels. Nifedipine abolished myogenic contraction and the increase in the fluorescence ratio. PKC inhibitors (H7 and staurosporine) abolished myogenic contraction but did not depress the increase in the fluorescence ratio. In mesenteric small arteries, myogenic contraction was insignificant in control vessels. A relatively low dose of PKC activator (4.4 +/- 1.4 nmol/l) elicited myogenic contraction, but a higher dose (21 +/- 6 nmol/l) depressed it. Thus the cytosolic Ca2+ increase and PKC activity may cooperatively act on the myogenic contraction of skeletal muscle small arteries. The activity of PKC should play an important role in myogenic contraction of rat small arteries.
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