We show by confocal immunofluorescence microscopy that the water channel protein aquaporin-1, not previously identified within cardiomyocytes, localizes at 20 and 37°C to rat cardiomyocyte sarcolemmal caveolar membrane and subsarcolemmal cytoplasm of primary atrial myocyte cultures, dissociated atrial and ventricular myocytes, and in situ cardiomyocytes of atrial and ventricular frozen sections. Confocal immunofluorescence microscopy shows that the normal in situ colocalization of the quasi-muscle-specific caveolar coating protein caveolin-3 with aquaporin-1 is reversibly disrupted by exposing in situ atrial or ventricular myocytes to physiological saline made hypertonic by adding 150 mM sucrose or 75 mM NaCl to isotonic physiological saline. This causes caveolae to close off from the interstitium and swell, while aquaporin-1 is internalized reversibly. At 4°C aquaporin-1 does not colocalize with caveolin-3. We suggest that 1) in vivo, under near-isotonic conditions, caveolae may alternate frequently between brief open and closed-off states; 2) aquaporin-1-caveolin-3 colocalization may be energy dependent; and 3) while closed off from the interstitium, each caveola transiently functions as an osmometer that experiences, monitors, and reacts to net water flow from or into the subcaveolar cytosol of the myocyte.

Sucrose-density flotation analysis of Triton-insoluble membrane domains isolated from highly purified sheep ventricular sarcolemma revealed the presence of two major 120- and 100-kDa proteins. Both species migrated in two-dimensional isoelectric focussing/SDS gels with an apparent pI of approximately 4.3, suggesting that they might be related. Microsequence analysis of peptides derived from the 100-kDa protein yielded amino acid sequences with high homology to T-cadherin, a truncated cadherin lacking a cytoplasmic domain. The similarity was confirmed using antibodies to chicken T-cadherin that reacted with both proteins on immunoblots. T-cadherin was released from the detergent-insoluble sarcolemmal fraction by phospholipase C treatment indicating that it is linked to the membrane by a glycophosphoinositol anchor. T-cadherin could be ADP-ribosylated by a transferase that was also present in the caveolin-enriched Triton-insoluble fraction. T-cadherin-containing membrane fragments cofractionated on sucrose gradients with caveolin-3, a marker protein for myocyte caveolae. However, immunopurified caveolin-3-containing membranes contained no associated T-cadherin. Immunocytochemical analysis of cultured rat atrial myocytes revealed that T-cadherin and caveolin have related but nonoverlapping staining patterns. These results suggest that T-cadherin is a major glycophosphoinositol-linked protein in cardiac myocytes and that it may be located in plasma membrane "rafts" distinct from but possibly adjacent to caveolae.

We have previously shown that atrial natriuretic peptide (ANP) is present in caveolae of in situ rat atrial myocytes. To investigate whether intracaveolar ANP of rat atrial myocytes exists within caveolae bound to type B ANP receptors (ANP-RB, a guanylyl cyclase), we have used confocal immunofluorescence microscopy applied to primary cultures of atrial myocytes from adult rats and to freshly dissociated rat atrial myocytes (not cultured). These experimental designs tested whether atrial myocyte ANP-RB colocalizes at the plasmalemma and elsewhere in the cell with the muscle-specific isoform of the caveolar coating protein caveolin-3, and with a fraction of cellular ANP. The experiments showed that cellular caveolin-3, a fraction of cellular ANP-RB, and a fraction of cellular ANP colocalize at the plasmalemma of cultured atrial myocytes and of freshly dissociated atrial myocytes. The observations support the hypothesis that in rat atrial myocytes, intracaveolar ANP is bound to ANP-RB, a protein whose cytosolic amino acid sequences are known to encode guanylyl cyclase activity. We suggest that among the (probably multiple) effects of the cGMP thus generated in the cytoplasmic microdomain underlying atrial myocyte caveolae may be the activation of cGMP-dependent protein kinase, which would thereby inhibit plasma membrane Ca2+ channel activity and contribute to a negative inotropic effect of ANP.

In isolated rat left ventricles perfused at 37 degrees C with control, Ca2+-loading, and Ca2+-depleting solutions (pH 7.3-7.4), we have investigated freeze-fractured gap junctional membrane by three quantitative techniques designed to correlate changes in junctional permeability with changes in membrane ultrastructure, i.e., 1) optical diffraction, 2) direct measurement of center-to-center spacings and particle diameters, and 3) statistical analysis of the spatial distribution of P-face particles based on analysis of nearest neighbor center-to-center distances. Junctions fixed either with glutaraldehyde or by quick freezing were compact, with closely packed rather than dispersed membrane particles even in the permeable state. Analysis of variance for all three methods indicated that replication was a major variability source limiting structural discrimination. Discrimination between random, regular, and clustered distributions depended critically on particle diameter and particle density. The results differ from published data of others on mammalian ventricular gap junctions and from measurements by our laboratory on sheep cardiac Purkinje fibers (J. Ultrastruct. Res. 75: 195-204, 1981).

We assessed the cellular localization and relative concentration of the C-type natriuretic peptide (CNP) guanylate cyclase-B (GC-B) receptor in the adult rat heart ventricle by several techniques. In frozen sections of the ventricle, anti-receptor antibody stained the vasculature and cells interstitial to myocytes, but not the myocytes themselves. The same antibody detected GC-B in immunoblots of protein extracts of nonmyocytes, but not myocytes and recognized an equivalent protein in extracts of cultured cardiac fibroblasts, but not A7r5 rat smooth muscle cells. In functional assays, CNP-induced cGMP accumulation per milligram cell protein was an order of magnitude greater in cultured cardiac fibroblasts than in A7r5 smooth muscle cells and two orders of magnitude greater than in freshly isolated cardiac myocytes. Modulation of cGMP accumulation by phosphodiesterases (PDEs) was cell specific as determined by antagonist pharmacological profiles, PDE1 in fibroblasts, PDE2 in A7r5 cells, and PDE3 in myocytes, suggesting that significant but low-level cGMP response to CNP measured in heart myocytes is not due to nonmyocyte contamination. Fibroblasts of cardiac origin do not show an interactive relationship between receptor responsiveness to CNP, cGMP levels, and proliferation-related mitogen-activated signal transduction pathways. Whereas previous reports suggest CNP exerts significant effects in neonatal rat cardiomyocytes, our results suggest that fibroblasts are likely the most responsive cell type (cGMP production) in the adult rat heart. cardiomyocytes; fibroblasts; smooth muscle; guanylate cyclase C-TYPE NATRIURETIC PEPTIDE (CNP) is a member of the family of NP hormones that also includes atrial NP (ANP) and brain NP (BNP) (26). ANP is synthesized in cardiac myocytes, where it is secreted constitutively in both the ventricle and atrium as well as in a regulated manner from granules in the atrium. First discovered in the brain, CNP has also been found in other tissues as well, e.g., in bone, reproductive tissue, and heart tissue (6, 22). CNP is secreted by endothelial cells in the heart (25), but its role in myocardial function is much less clear than that of ANP. There are presently three known NP receptors (NPR): NPR-A, -B, and -C. In an anomaly of nomenclature, NPR-A binds ANP and BNP in the nanomolar range and CNP in the micromolar range, whereas NPR-B binds CNP in the nanomolar range but binds ANP and BNP only in the micromolar range (17). Both of these transmembrane receptors express guanylyl cyclase (GC) activity in their cytoplasmic domains and are also known as GC-A and GC-B (27). NPR-C, found most abundantly in the kidney, has a truncated cytoplasmic tail, has no cyclase activity, and is believed to be involved predominantly in NP clearance (21), although a role in G i -dependent signaling has recently been proposed (15).mRNA for all three receptor types was detected by RT-PCR in myocytes isolated from the adult rat ventricle, but expression at the protein level was convincingly shown for GC-A, but n...