Intravital microscopy was used to study the effect of endothelium-derived relaxing factor (EDRF) on microvascular adrenoceptor sensitivity in rat cremaster skeletal muscle. NG-Monomethyl L-arginine (L-NMMA, 1-300 ,uM), an inhibitor of EDRF formation, produced concentration-dependent constriction of arterioles and venules. When an intermediate amount of a-l versus a2-adrenoceptor tone was first produced with bath-added norepinephrine (NE) in the presence of rauwolscine or prazosin, L-NMMA caused constriction with greater potency and efficacy during aY2 than during crl tone. During localized arl or aY2 constriction along an arteriole by perivascular micropipette suffusion of NE in the presence of rauwolscine or prazosin, again, bath-added L-NMMA produced constriction with greater potency during a2 than during arl constriction. Like L-NMMA, disruption of EDRF release by microembolization caused baseline arteriole constriction and selectively increased aY2 sensitivity 75-fold. Although these findings support the hypothesis that endothelial cells possess ar2-adrenoceptors that promote EDRF release, a greater susceptibility of a2 than arl constriction to EDRF inhibition could also account for the results. In support of this latter possibility, a2 constriction was approximately 50-fold more susceptible than al constriction to inhibition by the EDRF-like nitrodilator nitroprusside. The similarity in magnitude of this difference in sensitivity with the difference obtained in the embolization experiments does not support the hypothesis that microvascular endothelial cells in skeletal muscle possess EDRF-promoting av2-adrenoceptors. However, these data do suggest that endogenous EDRF release modulates basal arteriole and venule tone and that aY2-adrenoceptor constriction is more sensitive than a, constriction to inhibition by EDRF. (Circulation Research 1992;71:188-200) KEY WoRDs * endothelium-derived relaxing factor * a-adrenergic receptors * arterioles venules * NG_monomethyl L-arginine * endothelial cells * microcirculation Since the discovery1 that acetylcholine (ACh) can stimulate endothelial cells to release a potent vasodilator substance-endothelium-derived relaxing factor (EDRF) -that is nitric oxide or a nitric oxide adduct,23 much has been learned about its synthesis, metabolism, release, and mode of relaxant action on vascular smooth muscle.45 On the other hand, the significance of EDRF in vascular regulation is not as well understood. The dilator properties of a number of vasoactive substances, e.g., ACh, bradykinin, and histamine, are mediated partly or entirely by EDRF.5 Also, flow-mediated dilation appears to involve a shearstress-induced increase in EDRF release.67 In certain8,9 but not all10-12 vessels, myogenic constriction may be mediated in part by endothelial cell vasoactive factors including EDRF. Inhibition of EDRF synthesis by substituted L-arginine compounds that serve as substrate inhibitors of EDRF synthetase13 produces increases in blood pressure and vascular resistance in

The relative contribution of postjunctional Gi-and a2-adrenoceptors to constriction of microvessels was examined during sympathetic nerve stimulation and sympathetic escape (difference between peak and steady-state constriction). Large arterioles (120±4 ,um control diameter) and venules (174±6 ,um) and small arterioles (13+±4 ,um) were examined in rat cremaster skeletal muscle during stimulation of the cremaster efferent innervation (decentralized lumbar sympathetic chain, 0.5-16 Hz, 2-minute train). The muscle was suspended in a tissue bath, and diameter was measured with intravital microscopy. Frequency-response curves were obtained after vehicle (prazosin or rauwolscine) was added to the bath. In large arterioles, prazosin (10-7 M) significantly attenuated constriction by 60-80%; a fivefold higher concentration had no additional effect. In contrast, rauwolscine (1 to 5 x 10-7 M) had no effect. Venules evidenced minimal response to nerve stimulation. In small arterioles, rauwolscine (5 x 10-7 M) significantly attenuated constriction by 50-60%, while prazosin (10-M) had no effect. These data suggest that for large arterioles, which are known to possess both receptors, a1l-adrenoceptors are preferentially stimulated by nerve-released norepinephrine. In contrast, sympathetic constriction of small arterioles is mediated by a2-adrenoceptors. Compared with large arterioles, small arterioles exhibited greater peak and steady-state constriction at all frequencies, with maximal responses achieved over the 0.5-4 Hz range. Large arterioles exhibit graded constriction over the entire frequency range. Sympathetic escape exhibited a small, negatively correlated frequency dependence for large arterioles, tended to be greater for small arterioles, and was more evident in large arterioles during aV2-adrenoceptor constriction at low-frequency stimulation. This distinct neural control of large resistance vessels by a1-adrenoceptors and small terminal arterioles by a2-adrenoceptors may allow neurogenic regulation of these vessel segments to be differentially susceptible to modulation by other extrinsic and intrinsic vasoactive controls that preferentially interact with al-and av2-adrenergic contractile mechanisms. (Circulation Research 1991;68:232-244) V ascular smooth muscle can possess al-and a2-adrenoceptors that both mediate contraction.1-4 Whether postjunctional a1-and a2-adrenoceptors subserve distinct physiological roles is unknown. Several earlier studies of whole animals or regional vascular beds revealed preferential blockade by a2-antagonists of pressor responses to blood-borne norepinephrine (NE), whereas a1-antagonists prefer-

The interaction of alpha 1- and alpha 2-adrenoceptor constriction of isolated rat cremaster small arteries (mean diameter +/- SEM, 114 +/- 5 microns) with the myogenic mechanism was examined with in vitro videomicroscopy. Microdissected vessels were double-cannulated with glass micropipets in a tissue bath, and intraluminal pressure was set at 65 mm Hg baseline pressure. Norepinephrine (NE) concentration-response curves were obtained alone and in the presence of prazosin or rauwolscine to establish the concentration of NE required to selectively stimulate alpha 1- or alpha 2-adrenoceptors. A bimodal response was produced by NE alone, indicating interaction with more than one receptor population. Rauwolscine and prazosin each produced dextral displacement at low (< 0.1 microM) NE concentrations. However, while prazosin exhibited competitive antagonism, rauwolscine did not antagonize NE at concentrations > or = EC50 value. Thus, both alpha 1- and alpha 2-adrenoceptors mediate constriction at low NE concentrations, but only alpha 1-receptors contributed to constriction at intermediate and high concentrations. These data are in contrast to previous findings for the same vessels studied in vivo. Diameter responses to increases and decreases in transmural pressure from baseline were examined in the relaxed (passive) state with nitroprusside present, during spontaneous intrinsic tone, and during induced alpha 1- or alpha 2-tone (EC20 concentration of NE plus rauwolscine or prazosin). Myogenic constriction during increases in lumen pressure and myogenic inhibition of tone during decreases in pressure were greatest during alpha 2-tone, less during intrinsic tone and least during alpha 1-tone.(ABSTRACT TRUNCATED AT 250 WORDS)

alpha 2-Adrenoceptor but not alpha 1-adrenoceptor constriction of arterioles is selectively inhibited by tissue acidosis, ischemia, and increased metabolic rate. To further examine neural-local interactions, we studied the effect of adenosine receptor stimulation on alpha 1- or alpha 2-adrenoceptor constriction. Intravital microscopy was used to study large arterioles (133 +/- 3 microns diam; mean +/- SE), small arterioles (16 +/- 1 microns), and large venules (178 +/- 3 microns) of rat cremaster skeletal muscle. Concentration-response (diameter change) curves were obtained for bath-added norepinephrine in the presence of either rauwolscine or prazosin to provide selective alpha 1- and alpha 2-constriction, respectively. The adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine (2.24 x 10(-8) M) significantly attenuated both alpha 1- and alpha 2-constriction by 5- to 20-fold; alpha 1-constriction was three- to fourfold more sensitive than alpha 2-constriction. Similar inhibitory effects were obtained with adenosine (2.24 x 10(-6) M). The adenosine receptor antagonist 8-[4-[N(2-aminoethyl)carbamoylmethoxy]phenyl]-1,3-dipropylxanthine (0.7 microM) reversed the inhibitory effect of adenosine, which implicates extracellular A2 adenosine receptors. Intrinsic tone in large vessels was unaffected by adenosine receptor stimulation but was completely inhibited in small arterioles. These findings suggest that both alpha 2- and especially alpha 1-adrenoceptor constriction and intrinsic tone (of small but not large arterioles) are inhibited by physiologically relevant concentrations of adenosine.

Background-Extravascular smooth muscle cells are often observed in the endocardium of human failing hearts. Here, we characterized the phenotype of those cells and investigated their physiological significance. Methods and Results-We examined left ventricular biopsy specimens obtained from 44 patients with dilated cardiomyopathy and 6 nonfailing hearts. In Masson trichrome-stained histological preparations, bundles of smooth muscle cells were seen localized in the endocardium in 23 of the 44 specimens (none of the 6 controls). These cells were immunopositive for α-smooth muscle actin, type 2 smooth muscle myosin, desmin, and calponin, but were negative for embryonic smooth muscle myosin, vimentin, fibronectin, and periostin. This profile is indicative of a late differentiation (contractile) smooth muscle phenotype. Electron microscopy confirmed that phenotype, revealing the cells to contain abundant myofilaments with dense bodies but little rough endoplasmic reticulum or Golgi apparatus.