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PubMed1992

Colocalization of peptide and glucocorticoid receptor immunoreactivities in rat central amygdaloid nucleus.

The central amygdaloid nucleus (ACe) is part of the amygdaloid complex that participates in adrenocorticotrophin secretion, stress-related reactions and behavioral functions. The ACe contains numerous glucocorticoid receptor (GR)-immunoreactive (IR) neurons, and in addition it has been shown to contain several neuropeptide-IR somata and nerve terminals. In order to study the relationship between the GR- and neuropeptide-IR structures we mapped the distribution of GR-like immunoreactivity (LI) in amygdaloid complex and colocalized the neuropeptide- and GR-LIs in the ACe. In the amygdaloid complex the central, medial and cortical nuclei contained a high number of GR-IR neurons, whereas a moderate number of GR-IR neurons were observed in the basolateral and basomedial nuclei. Only a few GR-IR neurons were seen in the lateral nucleus. In the ACe, the majority of corticotrophin-releasing factor (CRF)-, met-enkephalin (met-ENK)-, neurotensin (NT)- and somatostatin (SOM)-IR neurons contained also GR-IR. About half of the substance P (SP)-IR neurons were seen to contain GR-IR, whereas only some of the few vasoactive intestinal polypeptide and cholecystokinin-IR neurons showed GR-LI. Nerve terminals containing calcitonin gene-related peptide and the above mentioned peptides were seen in close contact with the GR-IR neurons. These results suggest that the glucocorticoids may modulate directly the neurotransmitter synthesis of the CRF-, met-ENK, NT-, SOM- and SP-IR cells in the ACe.

Amygdala↗

PubMed1992

Colocalization of somatostatin receptors and growth hormone-releasing factor immunoreactivity in neurons of the rat arcuate nucleus.

Recent studies from our group have demonstrated an association of [125I]-labeled somatostatin (SRIF)-binding sites with a subpopulation of arcuate (ARC) neurons. The distribution of these cells was similar to that of growth hormone-releasing factor (GRF)-immunoreactive neurons, which led us to propose that at least some SRIF receptors may be directly localized to GRF-containing cells. To test this hypothesis, we have visualized radiolabeled SRIF-binding sites and GRF immunoreactivity (ir) in adjacent sections of the hypothalamus, by combined radioautography and immunohistochemistry. Adult male rats were sacrificed by decapitation and the brains were rapidly frozen and serially sectioned on a cryostat. Fifteen pairs of adjacent 6-microns-thick sections, taken at 100-microns intervals through the rostrocaudal extent of the ARC nucleus, were alternately processed for [125I]-SRIF radioautography and GRF immunohistochemistry. GRF-ir and [125I]-SRIF-labeled cells were mapped at each level and quantified with the aid of a camera lucida. The maps were subsequently superimposed to determine the extent of [125I]-SRIF/GRF-ir colocalization. GRF-ir perikarya [13.2 +/- 4.4 (mean +/- SE) cells per section] were mainly localized in the ventrolateral portion of the ARC nucleus and predominated within the caudal-most tier. [125I]-SRIF-labeled cells (35.6 +/- 6.5 cells per section) were more numerous, more evenly distributed, and extended further rostrally and caudally than GRF-ir cells. Superimposition of the camera lucida maps indicated that, overall, 33.5 +/- 10.8% of the GRF-ir cells were labeled with [125I]-SRIF in adjacent sections.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

Colocalization of corticotropin-releasing factor and vasopressin in the paraventricular nucleus of the human hypothalamus.

The anatomical relationship between corticotropin-releasing factor (CRF)-containing cells and arginine vasopressin (AVP)-containing cells in the human hypothalamus was investigated by immunocytochemistry. In the paraventricular nucleus of the hypothalamus (PVH), CRF-like immunoreactivity (CRF-LI) was present exclusively in parvocellular cells, while AVP-like immunoreactivity (AVP-LI) was present in both parvocellular and magnocellular cells. No CRF-immunoreactive neurons were observed in the supraoptic nucleus. All CRF-immunoreactive parvocellular cells in the PVH were also AVP immunoreactive. We confirmed the presence of AVP in the CRF-immunoreactive cells by using two kinds of anti-AVP antisera, one of which recognized the side chain of AVP while the other recognized the ring structure of AVP. Colocalization of CRF-LI and AVP-LI was observed not only in the same perikarya but also in the same nerve fibers of parvocellular cells. The present results raise the possibility that AVP and CRF may be secreted together into the human portal circulation.

Adolescent↗

PubMed1996

Colocalization of progesterone receptor and somatostatin immunoreactivities in the hypothalamus of the male and female guinea pig.

A double-label immunofluorescence technique was used to determine whether progesterone receptor (PR)-containing neurons in the preoptic area and hypothalamus also contain somatostatin (SOM) in both the male and female guinea pig. Animals were gonadectomized, primed by estradiol to induce PR and injected intracerebroventricularly with colchicine to visualize SOM-immunoreactive (SOM-IR) neurons. The only sites of significant overlap between the two immunoreactivities were the medial preoptic nucleus, the periventricular preoptic and hypothalamic regions, the arcuate nucleus (Ar) and the ventrolateral nucleus (VL). No sex differences were detected at this level. In the preoptic area and the periventricular regions, no SOM-IR neurons were shown to have PR. In the Ar, only very few SOM-IR perikarya were found to be also PR-IR. SOM varicosities appeared in close proximity to neurons with PR-containing nuclei. Within the VL, in the female as well as in the male, many SOM-IR cells were also IR for PR. This colocalization persisted throughout the rostrocaudal extent of the nucleus but our quantification revealed a significant sex difference in the percentage of PR-IR neurons with SOM in the caudal VL. These results provide neuroanatomical evidence that progesterone may exert its effect directly upon more than one third of SOM-synthesizing cells in the medial and caudal regions of VL, a site which plays a key role in the control of sexual behavior.

Animals↗

PubMed1991

Cellular and subcellular localization of progastricsin in calf fundic mucosa: colocalization with pepsinogen and prochymosin.

The presence of gastricsin in bovine abomasal juice has been reported previously, but its exact site of origin has not yet been established. Specific polyclonal antibodies were used in the peroxidase-antiperoxidase method or the protein A/gold technique to label cells producing progastricsin. This immunocytolocalization was correlated with that of pepsinogen and prochymosin using specific polyclonal antibodies against those zymogens. The present study clearly established that progastricsin was located exclusively in chief, mucous neck, transitional mucous neck/chief, foveolar epithelial and surface epithelial cells of the calf fundic mucosa. Furthermore, progastricsin was found to be colocalized with pepsinogen and prochymosin in the same secretory granules of these cells. Progastricsin was not observed in parietal, gastric endocrine and undifferentiated neck cells.

Abomasum↗

PubMed2004

C-peptide colocalizes with macrophages in early arteriosclerotic lesions of diabetic subjects and induces monocyte chemotaxis in vitro.

OBJECTIVE: Increased levels of C-peptide, a cleavage product of proinsulin, circulate in patients with insulin resistance and early type 2 diabetes, a high-risk population for the development of a diffuse and extensive pattern of arteriosclerosis. This study tested the hypothesis that C-peptide might participate in atherogenesis in these patients. METHOD AND RESULTS: We demonstrate significantly higher intimal C-peptide deposition in thoracic aorta specimens from young diabetic subjects compared with matched nondiabetic controls as determined by immunohistochemical staining. C-peptide colocalized with monocytes/macrophages in the arterial intima of artery specimen from diabetic subjects. In vitro, C-peptide stimulated monocyte chemotaxis in a concentration-dependent manner with a maximal 2.3+/-0.4-fold increase at 1 nmol/L C-peptide. Pertussis toxin, wortmannin, and LY294002 inhibited C-peptide-induced monocyte chemotaxis, suggesting the involvement of pertussis toxin-sensitive G-proteins as well as a phosphoinositide 3-kinase (PI3K)-dependent mechanism. In addition, C-peptide treatment activated PI3K in human monocytes, as demonstrated by PI3K activity assays. CONCLUSIONS: C-peptide accumulated in the vessel wall in early atherogenesis in diabetic subjects and may promote monocyte migration into developing lesions. These data support the hypothesis that C-peptide may play an active role in atherogenesis in diabetic patients and suggest a new mechanism for accelerated arterial disease in diabetes.

Adolescent↗

PubMed1997

Immunohistochemical colocalization of the terminal complex of human complement and smooth muscle cell alpha-actin in early atherosclerotic lesions.

There is substantial evidence that activated components of the complement cascade are present in atherosclerotic lesions, and it was suggested some years ago that smooth muscle cells may be an important target of complement attack by the terminal components of the cascade, C5b-9, also called the membrane attack complex. Recent in vitro studies have shown that assembly of membrane attack complex on smooth muscle cells leads to the release of monocyte chemotactic protein-1, and, if this were to occur in vivo, then it could be responsible for the recruitment of monocytes into the lesion. In this study we have investigated the localization of C5b-9 in early atherosclerotic lesions of human coronary arteries, collected from autopsies, by immunohistochemical staining, C5b-9 was found to colocalize widely with smooth muscle cell alpha-actin, but not with intact macrophages, thus supporting the hypothesis that interaction of complement with smooth muscle cells may indeed be important in atherogenesis.

Actins↗

PubMed1997

Tissue factor pathway inhibitor in endothelial cells colocalizes with glycolipid microdomains/caveolae. Regulatory mechanism(s) of the anticoagulant properties of the endothelium.

Tissue factor pathway inhibitor (TFPI), the main downregulator of the procoagulant activity of tissue factor.factor VIIa complex, locates in human endothelial cells (EC) in culture as well-defined clusters uniformly distributed both on the cell surface and intracellularly. We here demonstrate by immunofluorescence that TFPI colocalizes in EC with caveolin, urokinase-type plasminogen activator receptor, and glycosphingolipids. The localization of TFPI in caveolae in resting endothelium is proved by double immunogold electron microscopy for TFPI and caveolin. After ultracentrifugation of rat lung or EC homogenates through density gradients of Nycodenz, TFPI was highly enriched at densities of 1.05 to 1.08 g/mL, together with caveolin and alkaline phosphatase. By ELISA, more than half of the cellular TFPI was detected in Triton X-100-insoluble extracts of EC. TFPI incorporates [1-3H]ethanolamine and is cleaved from the cell surface by phosphatidylinositol-phospholipase C, indicating a specific glycosylphosphatidylinositol-anchorage mechanism for TFPI in the plasma membrane. Clustering of TFPI and its localization in caveolae are dependent on the presence of cholesterol in the membrane. Agonist-induced stimulation of EC caused marked changes of distribution for both TFPI and caveolin at subcellular level, with subsequent increase of the cell surface-associated inhibitory activity toward tissue factor.factor VIIa. Our findings suggest that, beside their function in transcytosis, potocytosis, cell surface proteolysis, and regulation of signal transduction, caveolae also play a direct role in the regulation of EC anticoagulant properties.

Animals↗

PubMed1998

Phospholipase A2 type II binds to extracellular matrix biglycan: modulation of its activity on LDL by colocalization in glycosaminoglycan matrixes.

We recently reported the presence of secretory, nonpancreatic phospholipase A2 type II (snpPLA2; EC 3.1.1.4) in human atherosclerotic arteries (Hurt-Camejo et al, Arterioscler Thromb Vasc Biol. 1997;17:300-309). SnpPLA2 may generate the proinflammatory products lysophospholipids and free fatty acids, thus contributing to atherogenesis when acting on low density lipoproteins (LDLs) retained in the arterial wall. Immunohistochemical studies showed that smooth muscle cells (SMCs) in human arterial tissue are the main sources of snpPLA2. In cultures of human arterial SMCs, snpPLA2 interacts with versican and smaller heparan/chondroitin sulfate proteoglycans (PGs) secreted as soluble components into the medium. In the present study, we investigated the binding of snpPLA2 to extracellular matrix (ECM) PGs produced by SMCs. The results show that snpPLA2 can bind to the ECM at physiological salt concentrations. ECM-bound snpPLA2 was active, hydrolyzing phosphatidylcholine-containing micelles. Soluble chondroitin-6-sulfate at concentrations >1 micromol/L, but not heparin or heparan sulfate, was able to release ECM-bound snpPLA2. The PG mainly involved in the binding of snpPLA2 was identified as biglycan. Perlecan was also present in the ECM synthesized by SMCs, but it contributed less to the binding of snpPLA2. Experiments with immobilized glycosaminoglycans indicated that snpPLA2 hydrolyzed 7-fold more LDL phospholipids when the lipoprotein and the enzyme were colocalized in a matrix with chondroitin-6-sulfate compared with one with heparin. These data suggest that retention of snpPLA2 in ECMs of different composition may modulate the enzymatic activity of snpPLA2 toward LDL. The results presented in this work support the hypothesis of the potential contribution of snpPLA2 to atherosclerosis.

Animals↗

PubMed1998

Immunohistochemical demonstration of enzymatically modified human LDL and its colocalization with the terminal complement complex in the early atherosclerotic lesion.

Treatment of low density lipoprotein (LDL) with degrading enzymes transforms the molecule to a moiety that is micromorphologically indistinguishable from lipoproteinaceous particles that are present in atherosclerotic plaques, and enzymatically modified LDL (E-LDL), but not oxidized LDL (ox-LDL), spontaneously activates the alternative complement pathway, as do lesion lipoprotein derivatives. Furthermore, because E-LDL is a potent inducer of macrophage foam cell formation, we propose that enzymatic degradation may be the key process that renders LDL atherogenic. In this article, we report the production of two murine monoclonal antibodies recognizing cryptic epitopes in human apolipoprotein B that become exposed after enzymatic attack on LDL. One antibody reacted with LDL after single treatment with trypsin, whereas recognition by the second antibody required combined treatment of LDL with trypsin and cholesterol esterase. In ELISAs, both antibodies reacted with E-LDL produced in vitro and with lesion complement activator derived from human atherosclerotic plaques, but they were unreactive with native LDL or ox-LDL. The antibodies stained E-LDL, but not native LDL or ox-LDL, that had been artificially injected into arterial vessel walls. With the use of these antibodies, we have demonstrated that early human atherosclerotic coronary lesions obtained at autopsy as well as lesions examined in freshly explanted hearts always contain extensive extracellular deposits of E-LDL. Terminal complement complexes, detected with a monoclonal antibody specific for a C5b-9 neoepitope, colocalized with E-LDL within the intima, which is compatible with the proposal that subendothelially deposited LDL is enzymatically transformed to a complement activator at the earliest stages in lesion development.

Antibodies, Monoclonal↗

PubMed2000

Colocalization of thrombin, PAI-1, and vitronectin in the atherosclerotic vessel wall: A potential regulatory mechanism of thrombin activity by PAI-1/vitronectin complexes.

The serine protease thrombin is a mitogen for vascular smooth muscle cells. To that end, thrombin cleaves the surface-exposed, protease-activated receptor type 1 (PAR-1), resulting in signal transduction and ultimately, proliferation of these cells. Regulation of thrombin activity in the human atherosclerotic vessel wall has not been studied in great detail, conceivably because the traditional plasma thrombin inhibitor, anti-thrombin III, is not encountered at this location. By using immunofluorescence confocal microscopy, we demonstrate that the antigens of thrombin, plasminogen activator inhibitor 1 (PAI-1), and vitronectin (Vn) colocalize in human neointimal atherosclerotic arterial tissue. Furthermore, it is shown by in situ reverse zymography that these specimens harbor the active form of PAI-1, which is the only configuration of PAI-1 capable of complexing with Vn and inhibiting serine proteases, eg, thrombin. Two different criteria were used to establish that neointimal atherosclerotic material contains active alpha-thrombin, namely, its ability to bind to the thrombin inhibitor hirudin and to convert the thrombin-specific chromogenic substrate S2238. The latter activity could be fully prevented by preincubation with the thrombin-specific inhibitor, phenyl-prolyl-arginyl-chloromethyl ketone. The thrombin concentration measured by conversion of the chromogenic substrate was 7 to 12 nmol/L in the vascular specimens studied. This concentration range suffices to activate the PAR-1 receptor on vascular smooth muscle cells and to cause neointimal proliferation. It is concluded that the human atherosclerotic arterial vessel wall provides conditions that favor a regulatory mechanism of thrombin activity by PAI-1/Vn complexes.

Aorta↗

PubMed2001

Human coxsackie-adenovirus receptor is colocalized with integrins alpha(v)beta(3) and alpha(v)beta(5) on the cardiomyocyte sarcolemma and upregulated in dilated cardiomyopathy: implications for cardiotropic viral infections.

BACKGROUND: The coxsackievirus and adenovirus receptor (CAR) was identified as a common cellular receptor for both viruses, but its biological and pathogenic relevance is uncertain. Knowledge of CAR localization in the human cardiovascular system is limited but important with respect to CAR-dependent viral infections and gene transfer using CAR-dependent viral vectors. METHODS AND RESULTS: Explanted failing hearts from 13 patients (8 with dilated cardiomyopathy [DCM] and 5 with other heart diseases [non-DCM]) and normal donor hearts (n=7) were investigated for the expression levels and subcellular localization of CAR and the adenovirus coreceptors alpha(v)beta(3) and alpha(v)beta(5) integrins. CAR immunoreactivity was very low in normal and non-DCM hearts, whereas strong CAR signals occurred at the intercalated discs and sarcolemma in 5 of the 8 DCM hearts (62.5%); these strong signals colocalized with both integrins. In all hearts, CAR was detectable in subendothelial layers of the vessel wall, but not on the luminal endothelial surface, and on interstitial cells. Human CAR (hCAR) expressed in rat cardiomyocytes was targeted to cell-cell contacts, which resembled CAR localization in DCM hearts and resulted in 15-fold increased adenovirus uptake. CONCLUSIONS: Low hCAR abundance may render normal human myocardium resistant to CAR-dependent viruses, whereas re-expression of hCAR, such as that observed in DCM, may be a key determinant of cardiac susceptibility to viral infections. Asymmetric expression of hCAR in the vessel wall may be an important determinant of adenovirus tropism in humans. hCAR subcellular localization in human myocardium and hCAR targeting to cell-cell contacts in cardiomyocyte cultures suggest that hCAR may play a role in cell-cell contact formation.

Adenoviridae↗

PubMed1997

C-reactive protein colocalizes with complement in human hearts during acute myocardial infarction.

BACKGROUND: Rises in circulating C-reactive protein (CRP), the prototypical acute-phase protein in humans, correlate with clinical outcome in patients with myocardial ischemia and infarction. We hypothesized that these correlations might reflect active participation of CRP in the local inflammatory response ensuing in the jeopardized myocardium because on binding to a ligand, CRP is able to activate the classic pathway of complement, and in addition, complement activation has been shown to occur locally in infarcted myocardium. METHODS AND RESULTS: To verify our hypothesis, we investigated localization of CRP in relation to deposition of complement in tissue specimens of infarcted and healthy heart tissue obtained from 17 patients who had died after acute myocardial infarction. CRP was found to be deposited only in infarcted regions and not in normal-appearing areas of the myocardium, being colocalized with depositions of C4 and C3 activation fragments of the complement system. Deposition of CRP and complement in infarcted myocardium appeared to be time dependent, because it was found in all infarctions except for one of young age (< 12 hours old) and two of greater age (> 1 year old), whereas another tissue specimen of an infarct < 12 hours old showed only moderate but positive staining for both CRP and complement in comparison with older infarctions. CONCLUSIONS: We conclude that in humans, CRP may localize in infarcted heart tissue and suggest that this acute-phase protein promotes local complement activation, and hence tissue damage, in acute myocardial infarction.

Aged↗

PubMed1997

Colocalization of CPP-32 with apoptotic cells in human atherosclerotic plaques.

BACKGROUND: Apoptosis that has been reported in human atherosclerosis may contribute to the remodeling of atherosclerotic plaques. The identification of specific markers for apoptosis in these plaques would permit the development of specific therapeutic strategies to limit their progression. Cysteine protease CPP-32 is essential for apoptotic death in mammalian cells and appears to be an attractive candidate. METHODS AND RESULTS: We studied 12 atherosclerotic plaques from 12 patients who underwent carotid endarterectomy. Apoptosis was analyzed by in situ end labeling of fragmented DNA (TUNEL method) and corroborated by the presence of DNA fragmentation in agarose gel electrophoresis. CPP-32 was detected with the use of a specific monoclonal antibody, and its expression was compared with that of interleukin-1beta-converting enzyme (ICE). We showed that CPP-32 was highly expressed in 10 of 12 atherosclerotic plaques and that it colocalized with apoptotic cells. Expression of ICE generally paralleled that of CPP-32, but ICE was also detected in plaques negative for CPP-32 and showing no apoptosis. CONCLUSIONS: CPP-32 is highly expressed within human atherosclerotic plaques and is closely related to apoptosis. This finding suggests that CPP-32 may be the ICE-like enzyme responsible for apoptosis in human atherosclerosis and opens new perspectives for the development of therapeutic strategies to alter the progression of this disease.

Apoptosis↗

PubMed1998

Comparison of apolipoprotein and proteoglycan deposits in human coronary atherosclerotic plaques: colocalization of biglycan with apolipoproteins.

BACKGROUND: Because the content of specific proteoglycans and apolipoproteins is increased in atherosclerotic plaques and in vitro studies have suggested a role for proteoglycans in mediating plaque apolipoprotein (apo) retention, immunohistochemistry was performed to systematically examine the relative locations of proteoglycans and apolipoproteins in human atherosclerosis. METHODS AND RESULTS: The spatial relationships of versican, biglycan, and apoE were compared on 68 human coronary artery segments; apoA-I and apoB also were evaluated on an additional 20 segments. Nonatherosclerotic intima contained extensive deposits of versican, whereas deposits of apoE, apoB, and apoA-I were much less prevalent. In contrast, nearly all atherosclerotic segments contained substantial deposits of biglycan, apoE, apoA-I, and apoB. There was a high degree of colocalization of apoE and biglycan deposits. ApoA-I, the major apolipoprotein of HDL, and apoB also were detected in regions with apoE and biglycan deposition. Exceptions to the localization of biglycan with apolipoproteins were found in regions that lacked intact extracellular matrix because of necrosis or dense macrophage accumulation. In vitro studies demonstrated that biglycan binds apoE-containing but not apoE-free HDL and that biglycan also binds LDL. CONCLUSIONS: These results suggest that biglycan may bind apoE and apoB in atherosclerotic intima. They also raise the possibility that apoE may act as a "bridging" molecule that traps apoA-I-containing HDL in atherosclerotic intima. Taken together, these findings are consistent with the hypothesis that biglycan may contribute to the pathogenesis of atherosclerosis by trapping lipoproteins in the artery wall.

Apolipoprotein A-I↗

PubMed1999

Distinct patterns of transforming growth factor-beta isoform and receptor expression in human atherosclerotic lesions. Colocalization implicates TGF-beta in fibrofatty lesion development.

BACKGROUND: Some animal studies suggest that transforming growth factor-beta (TGF-beta) protects vessels from atherosclerosis by preventing intima formation, but others indicate a role in vessel proteoglycan accumulation and lipoprotein retention. To distinguish between these possibilities in humans, immunohistochemical studies were performed examining the coexpression of TGF-beta isoforms and the TGF-beta receptors ALK-5 and TbetaR-II in aorta during the various stages of atherosclerotic lesion development. METHODS AND RESULTS: The spatial relationships between TGF-beta1, TGF-beta3, ALK-5, and TbetaR-II expression were compared in aortic segments from 21 subjects. Nonatherosclerotic intima contained predominantly TGF-beta1, low concentrations of TbetaR-II, and barely detectable amounts of ALK-5. In contrast, fatty streaks/fibrofatty lesions contained high concentrations of both TGF-beta isoforms. Smooth muscle cells (SMCs), macrophages, and foam cells of macrophage and SMC origin contributed to these high levels. These lesions also contained high, colocalized concentrations of ALK-5 and TbetaR-II. Despite fibrous plaques containing TGF-beta1, its receptors were at detection limits. We found no evidence for truncated TbetaR-II expression in either normal intima or the various atherosclerotic lesions. CONCLUSIONS: TGF-beta appears to be most active in lipid-rich aortic intimal lesions. The findings support the hypothesis that TGF-beta contributes primarily to the pathogenesis of lipid-rich atherosclerotic lesions by stimulating the production of lipoprotein-trapping proteoglycans, inhibiting smooth muscle proliferation, and activating proteolytic mechanisms in macrophages.

Activin Receptors, Type I↗

PubMed1993

Dystrophin-glycoprotein complex and laminin colocalize to the sarcolemma and transverse tubules of cardiac muscle.

The expression and subcellular distribution of the dystrophin-glycoprotein complex and laminin were examined in cardiac muscle by immunoblot and immunofluorescence analysis of rabbit and sheep papillary muscle. The five dystrophin-associated proteins (DAPs), 156-DAG, 59-DAP, 50-DAG, 43-DAG, and 35-DAG, were identified in rabbit ventricular muscle and found to codistribute with dystrophin in both papillary myofibers and Purkinje fibers. The DAPs and dystrophin codistributed not only in the free surface sarcolemma but also in interior regions of the myofibers where T tubules are present. Neither the DAPs nor dystrophin were detected in intercalated discs, a specialized region of cardiac sarcolemma where neighboring myocardial cells are physically joined by cell-cell junctions. Similarly, in bundles of Purkinje fibers, which lack T tubules, DAPs and dystrophin were also found to codistribute at the free surface sarcolemma but were not detected either in the region of surface sarcolemma closely apposed to a neighboring Purkinje fiber or in interior regions of these myofibers. Comparison between the distribution of the dystrophin-glycoprotein complex and laminin showed that laminin codistributes with the components of this complex in both papillary myofibers and Purkinje fibers. These results are consistent with previous findings demonstrating that the extracellularly exposed 156-DAG (dystroglycan) of the skeletal muscle dystrophin-glycoprotein complex binds laminin, a component of the basement membrane. Although we demonstrate that DAPs, dystrophin, and laminin colocalize to the sarcolemma in rabbit and sheep papillary myofibers as they do in skeletal myofibers, the most striking difference between the subcellular distribution of these proteins in cardiac and skeletal muscle is that the dystrophin-glycoprotein complex and laminin also localize to regions of the fibers where T tubules are distributed in cardiac but not in skeletal muscle. These results imply that the protein composition and thus possibly some functions of T tubules in cardiac muscle are distinct from those of skeletal muscle.

Animals↗

PubMed1993

Multiple connexins colocalize in canine ventricular myocyte gap junctions.

We have recently shown that adult canine ventricular myocytes express three distinct gap junction channel proteins, connexin40 (Cx40), connexin43 (Cx43), and connexin45 (Cx45). These proteins have unique cytoplasmic domains that likely confer connexin-specific physiological properties. To determine whether the three distinct channel proteins are distributed in identical or different populations of gap junctions, we performed double-label immunofluorescence on disaggregated canine ventricular myocytes incubated simultaneously with a mouse monoclonal anti-Cx43 and affinity-purified polyclonal rabbit antibodies against Cx40 or Cx45. Analysis of double-labeled cardiac myocytes using laser scanning confocal microscopy revealed virtually identical patterns of immunoreactivity for both the Cx43/Cx40 and Cx43/Cx45 pairs. Double-label immunoelectron microscopy confirmed that ultrastructurally identified cardiac myocyte gap junctions contain multiple channel proteins. Thus, three channel proteins colocalize in canine cardiac myocyte gap junctions. The presence of multiple functionally distinct connexins suggests complex possibilities regarding the composition of individual channels and the regulation of intercellular coupling.

Animals↗