Development and initial evaluation of a novel method for assessing tissue-specific plasma free fatty acid utilization in vivo using (R)-2-bromopalmitate tracer.

We describe a method for assessing tissue-specific plasma free fatty acid (FFA) utilization in vivo using a non-beta-oxidizable FFA analog, [9,10-3H]-(R)-2-bromopalmitate (3H-R-BrP). Ideally 3H-R-BrP would be transported in plasma, taken up by tissues and activated by the enzyme acyl-CoA synthetase (ACS) like native FFA, but then 3H-labeled metabolites would be trapped. In vitro we found that 2-bromopalmitate and palmitate compete equivalently for the same ligand binding sites on albumin and intestinal fatty acid binding protein, and activation by ACS was stereoselective for the R-isomer. In vivo, oxidative and non-oxidative FFA metabolism was assessed in anesthetized Wistar rats by infusing, over 4 min, a mixture of 3H-R-BrP and [U-14C] palmitate (14C-palmitate). Indices of total FFA utilization (R*f) and incorporation into storage products (Rfs') were defined, based on tissue concentrations of 3H and 14C, respectively, 16 min after the start of tracer infusion. R*f, but not Rfs', was substantially increased in contracting (sciatic nerve stimulated) hindlimb muscles compared with contralateral non-contracting muscles. The contraction-induced increases in R*f were completely prevented by blockade of beta-oxidation with etomoxir. These results verify that 3H-R-BrP traces local total FFA utilization, including oxidative and non-oxidative metabolism. Separate estimates of the rates of loss of 3H activity indicated effective 3H metabolite retention in most tissues over a 16-min period, but appeared less effective in liver and heart. In conclusion, simultaneous use of 3H-R-BrP and [14C]palmitate tracers provides a new useful tool for in vivo studies of tissue-specific FFA transport, utilization and metabolic fate, especially in skeletal muscle and adipose tissue.

Vascular versus myocardial selectivity of calcium antagonists studied by concentration-time-effect relations.

The vascular versus myocardial selectivity of three structurally different calcium antagonists, felodipine, diltiazem, and verapamil, was studied in vitro. The model used was an isolated portal vein preparation and a paced (3 Hz) papillary muscle of the left ventricle of the rat, examined in the same organ bath. In previous experiments with the same tissues, used for screening and eventual selection of felodipine, it was found that the selectivity factors ranged as follows: felodipine much greater than nifedipine greater than diltiazem greater than verapamil = La3+. Since the effect of organic calcium antagonists is slow in onset, the present experiments were designed to determine the inhibitory potencies and the selectivity factor at equilibrium. This was possible by a computer-assisted collection of contractile force measurements and subsequent analysis of the results. In each experiment, one concentration only of one of the three calcium antagonists was given. The time-effect relation was determined for both tissues. After all experiments with a particular drug, when several concentrations had been administered, the hyperbolic concentration-effect relation was determined at various times of exposure. The ensuing pIC50 values, as related to time, for the vascular and the myocardial preparations, described by monoexponential curves and the corresponding potency values (pIC50), could be determined at equilibrium (t = infinity). It was concluded that the vascular over myocardial inhibitory selectivity was marked for felodipine (103), low for diltiazem (8.9), and none for verapamil (0.92).

Functional antagonism of noradrenaline responses by felodipine and other calcium antagonists in vascular smooth muscles.

Calcium antagonists inhibit vascular smooth muscle activity in a way that depends on the properties of the inhibitory agent, the type of effector tissue studied, and the applied means of activation. In the experiments described here, the phasically active portal vein of the rat and the essentially tonic aortae of the rat and the rabbit were examined. They were exposed to reduced external calcium ion concentration (from 2.5 mM to 0.6 and 0.2 mM) and to the structurally different calcium antagonists felodipine, verapamil, and diltiazem in "therapeutic" and 1,000-fold higher concentrations. The tissues were pretreated for 1 h with the intended [Ca2+]0 or the calcium antagonist concentration under study. Cumulative concentration-effect curves to noradrenaline (NA) were obtained in the control situation at reduced [Ca2+]0 and at two concentrations of a calcium antagonist. In the rat portal vein, low doses of all calcium antagonists suppressed all NA responses but caused no rightward shift of the concentration-effect curve. High doses of the calcium antagonist abolished portal vein spontaneous activity and reduced the maximum NA response to some 10% of control. In the rat portal vein, reduced [Ca2+] caused rightward shifts of the NA concentration-effect curve without (0.6 mM) or with (0.2 mM) reduced maximum NA responses. In the rat aorta, reduced [Ca2+]0 and the calcium antagonists caused progressive rightward shifts of the curves with progressive reductions in the maximum NA responses. In the rabbit aorta, there was no effect on NA responses with reduced [Ca2+]0 or of the calcium antagonists in "therapeutic" concentrations: verapamil and diltiazem, but not felodipine, caused significant effects in the 1,000-fold higher concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)

Angiotensin I converting enzyme activity in portal vein studied in normotensive rats and in models of primary and secondary hypertension.

The effects of angiotensin I (AT I), angiotensin II (AT II) and the activity of AT I converting enzyme (ACE) were investigated in isolated portal veins of normotensive rats (WKR and NCR) and of rats with primary (SHR) and secondary (one clip 2 kidney renal; RHR) hypertension. Based on ED50 values, the tissue sensitivity to AT II was slightly less in the portal veins of RHR than SHR, while the sensitivity of smooth muscle in NCR and WKR was similar to that of SHR. Angiotensin I induced contractions were inhibited by saralasin and by captopril, indicating presence of functionally active converting enzyme in the this vascular tissue. The local concentration of AT II formed from AT I, was evaluated based on AT II dose response curves and AT I response magnitude. The AT II formation was essentially similar in SHR, WKR and NCR and slightly enhanced in RHR. Inhibition of AT II formation with captopril and/or blockade of AT II receptors with saralasin failed to alter the spontaneous myogenic activity of the portal vein. Captopril reduced smooth muscle activity only in concentration several orders of magnitude greater than that needed to inhibit ACE. The concentration of captopril needed to inhibit AT I responses was similar in all strains of rats. It is concluded that AT II is rapidly formed in the portal vein due to local conversion of AT I. In the RHR portal vein the extent of AT I conversion is increased and the tissue sensitivity to AT II is decreased possibly due to mechanisms involved in the development of renovascular hypertension.

Contractile and morphological properties of the portal vein in spontaneously hypertensive and wistar-kyoto rats.

In perfusion experiments, previous investigations have shown an increased responsiveness of isolated vascular beds from spontaneously hypertensive rats (SHRs) compared with Wistar-Kyoto controls (WKYs). The results have suggested that there are differences in the arterial vessels but not in the venous vessels. Here we have tested the latter part of this hypothesis by comparing the contractile and morphological properties of isolated portal veins from 18 SHRs and 18 WKYs, all 3-4 months old. We found no differences between these veins as regards: (a) maximum force response; (b) active and passive force--length relationship; (c) sensitivity to exogenous noradrenaline without and with inhibition of neuronal uptake by cocaine, and (d) cross-sectional area of the longitudinal smooth muscle layer. There was, however, evidence for difference at the cellular level, for the active force per unit cell area was 17% lower in the SHR vessels compared with that in the WKY vessels. With this exception, the results indicate that the morphological and functional differences which have been reported by others for arterial vessels from SHRs and WKYs do not prevail in the portal vein taken from rats at 3-4 months of age.

Anti-hypertensive effect of metoprolol in spontaneously hypertensive rats.

1. Oral and intravenous administration of metoprolol to adult spontaneously hypertensive rats (SHR) with established hypertension lowered arterial blood pressure within 4 days of treatment. 2. Steady state plasma concentrations of metoprolol were similar to those of patients during anti-hypertensive treatment with this drug. 3. The neuroeffector function of portal veins of SHR treated orally for 14 days or intravenously for 4 days was not impaired when studied in vitro. This is in contrast to previous findings after long-term treatment. 4. It is concluded that the anti-hypertensive effect of metoprolol in SHR in many respects resembles that observed in patients. It is suggested that impairment of vasomotor nerve control may contribute to the anti-hypertensive effect of beta-adrenoreceptor antagonists.