K opioid receptors (K receptors) have been characterized in homogenates of guinea pig and rat brain under in vitro binding conditions. K receptors were labeled by using the tritiated prototypic K opioid ethylketocyclazocine under conditions in which ,u and 8 opioid binding was suppressed. In the case of guinea pig brain membranes, a single population of high-affinity K opioid receptor sites (K sites; Kd = 0.66 nM, BMMX = 80 fmol/mg of protein) was observed. In contrast, in the case ofrat brain, two populations of K sites were observedhigh-affinity sites at low density (Kd = 1.0 nM, Bm. = 16 fmol/mg of protein) and low-affinity sites at high density (Kd = 13 nM, Bin" = 111 fmol/mg of protein). To test the hypothesis that the high-and low-affinity K sites represent two distinct K receptor subtypes, a series of opioids were tested for their abilities to compete for binding to the two sites. U-69,593 and Cambridge 20 selectively displaced the high-affinity K site in both guinea pig and rat tissue, but were inactive at the rat-brain low-affinity site. Other K opioid drugs, including U-50,488, ethylketocyclazocine, bremazocine, cyclazocine, and dynorphin (1-17), competed for binding to both sites, but with different rank orders of potency. Quantitative light microscopy in vitro autoradiography was used to visualize the neuroanatomical pattern of K receptors in rat and guinea pig brain. The distribution patterns of the two K receptor subtypes of rat brain were clearly different. The pattern of rat high-affmiity K sites paralleled that of guinea pig in the caudate-putamen, midbrain, central gray substance of cerebrum, and substantia nigra; interspecies differences were apparent throughout most of the rest of the brain. Collectively, these data provide direct evidence for the presence of two K receptor subtypes; the U-69,593-sensitive, high-affinity K, site predominates in guinea pig brain, and the U-69,593-insensitive, low-affinity K2 site predominates in rat brain. Pharmacological studies have established that ketocyclazocine-like opioids produce their antinociceptive and unique sedative actions through an interaction with K receptors (2). These drugs effect a more pronounced sedation than do other opioids and have been evaluated as anesthetic agents. K opioid drugs neither suppress morphine abstinence nor induce abstinence in morphine-dependent monkeys (3). The endogenous opioid peptide dynorphin also interacts with high selectivity at K receptors.Evidence for a separate K receptor distinct from the morphine (A) and enkephalin (Enk; 8) receptors has been provided by pharmacological (2, 4), electrophysiological (5, 6), binding (4,7,8), and solubilization and purification (9)(10)(11) studies. In vitro autoradiography was used to visualize K receptors in rat (12) and guinea pig brain (13)

Left ventricular hypertrophy is based on cardiac myocyte growth. The hypertrophic process can be considered heterogeneous based on whether it also includes a remodeling and accumulation of fibrillar types I and III collagens that are responsible for impaired myocardial stiffness. In the heart, the messenger RNA (mRNA) for fibrillar collagen types I and III has been detected only in cardiac fibroblasts, whereas mRNA for basement membrane collagen type IV is present in both fibroblasts and myocytes. We studied the early and long-term expression of these collagenous proteins in rat myocardium after abdominal aortic banding with renal ischemia. Complementary DNA probes for rat pro-alpha 2 (I), mouse type III and mouse type IV collagens, and chicken beta-actin were used. Northern and dot blot analysis on total RNA extracted from left ventricular tissue indicated a sixfold increase in steady-state levels of mRNA for collagen type I on day 3 of abdominal aortic banding, which had declined to control levels by day 7 where it remained rather constant at 4 and 8 weeks. Type III collagen showed a similar pattern of gene expression after banding. mRNA levels for type IV collagen, on the other hand, were elevated on day 1 after banding, returning to control at day 7 and remaining constant. Actin mRNA levels also increased on day 1 of banding, followed by a rapid return to control levels. Monospecific antibody to types I and III collagens and immunofluorescent light microscopy on frozen sections of the myocardium revealed that at 1 week after banding, the distribution and density of these collagens were similar to those of control animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Cardiac fibroblasts are responsible for synthesis and deposition of fibrillar collagen types I and III. Transforming growth factor-,31 (TGF-j31) has been proved to increase collagen biosynthesis in various systems, both in vivo and in vitro. We have investigated the effect of TGF-P1 on collagen gene expression in cultured cardiac fibroblasts and have compared this effect with that of a mitogenic agent, phorbol myristate acetate (PMA). The regulation of collagen types I and III gene expression was examined by using cDNA probes to rat a£2 (I) and mouse a1 (III) procollagens. Quiescent cultured cardiac fibroblasts from rabbit heart were treated with TGF-(1 (10-15 ng/ml) and PMA (200 ng/ml). After 24 hours of treatment with TGF-f.1, the abundance of mRNA for pro-a2 (I) and pro-a1, (III) collagens was increased by 112% (p<0.001) and 97% (p=0.05), respectively, in treated fibroblasts compared with untreated cells. However, PMA-treated cells showed an opposite response: a 42% (p=0.01) decrease in mRNA levels for pro-a!2 (I) collagen was observed. Immunofluorescent staining of cardiac fibroblasts in culture with anti-type I collagen antibody showed that alterations in mRNA levels led to altered collagen synthesis: cellular collagen was relatively increased in TGF-f31-treated cells and significantly diminished in PMA-treated cells. The abundance of mRNA for pro-al (III) collagen was not affected by PMA treatment. Inereased collagen gene expression by TGF-f31 was abolished in the presence of cycloheximide, whereas the inhibitory effect of PMA on collagen type I mRNA did not change after addition of cycloheximide to the culture medium.

The extracellular matrix of the myocardium contains an elaborate structural matrix composed mainly of fibrillar types I and III collagen. This matrix is responsible for the support and alignment of myocytes and capillaries. Because of its alignment, location, configuration and tensile strength, relative to cardiac myocytes, the collagen matrix represents a major determinant of myocardial stiffness. Cardiac fibroblasts, not myocytes, contain the mRNA for these fibrillar collagens. In the hypertrophic remodeling of the myocardium that accompanies arterial hypertension, a progressive structural and biochemical remodeling of the matrix follows enhanced collagen gene expression. The resultant significant accumulation of collagen in the interstitium and around intramyocardial coronary arteries, or interstitial and perivascular fibrosis, represents a pathologic remodeling of the myocardium that compromises this normally efficient pump. This report reviews the structural nature, biosynthesis and degradation of collagen in the normal and hypertrophied myocardium. It suggests that interstitial heart disease, or the disproportionate growth of the extracellular matrix relative to myocyte hypertrophy, is an entity that merits greater understanding, particularly the factors regulating types I and III collagen gene expression and their degradation.