BACKGROUND:Caveolins (Cav) are structural proteins that insert into the plasma membrane to form caveolae that can bind molecules important in cardiac signal transduction and function. Cytochrome P450 epoxygenases can metabolize arachidonic acid to epoxyeicosatrienoic acids (EETs) which have known cardioprotective effects. Subsequent metabolism of EETs by soluble epoxide hydrolase reduces the protective effect.AIMS:(1) To assess the effect of ischemia-reperfusion injury on expression and subcellular localization of caveolins. (2) To study the effect of EETs on caveolins.METHODS:Hearts from soluble epoxide hydrolase null (KO) and littermate control (WT) mice were perfused in Langendorff mode and subjected to 20 minutes ischemia followed by 40 minutes reperfusion. Immunohistochemistry, immunoblot, and electron microscopy were performed to study localization of caveolins and changes in ultrastructure.RESULTS:In WT heart, Cav-1 and Cav-3 were present in cardiomyocyte and capillary endothelial cell at baseline. After ischemia, Cav-1 but not Cav-3, disappeared from cardiomyocyte; moreover, caveolae were absent and mitochondrial cristae were damaged. Improved postischemic functional recovery observed in KO or WT hearts treated with 11,12-EET corresponded to higher Cav-1 expression and maintained caveolae structure. In addition, KO mice preserved the Cav-1 signaling after ischemia that lost in WT mice.CONCLUSIONS:Taken together, our data suggest that ischemia-reperfusion injury causes loss of Cav-1 and caveolins, and EETs-mediated cardioprotection involves preservation of Cav-1.

Cytochrome P450 epoxygenases can metabolize arachidonic acid to epoxyeicosatrienoic acids (EETs) which have known cardioprotective effects. Subsequent metabolism of EETs by soluble epoxide hydrolase (sEH) reduces the protective effect. Caveolins (Cav-1, -2, and -3) are structural proteins that insert into the plasma membrane to form caveolae, which can bind molecules important in cardiac signal transduction and function. The objective of this study was to assess the role of Cav in EET mediated cardioprotection. Hearts from sEH null (KO) and littermate control (WT) mice were perfused in Langendorff mode and subjected to 20min ischemia followed by 40min reperfusion. Immunohistochemistry, immunoblot and electron microscopy were performed to study localization of Cav and changes in ultrastructure. In WT heart, cav-1 and cav-3 were present in cardiomyocyte (CM) and capillary endothelial cell at baseline. Following ischemia, cav-1 but not cav-3 disappeared from CM; moreover, caveolae were absent in WT hearts. Mitochondria and T-tubules were swollen, and cristae of mitochondria were damaged. Improved postischemic functional recovery observed in KO or WT hearts treated with 11,12-EET corresponded with higher cav-1 expression in plasma membrane and mitochondrial fractions. Taken together, our data suggest EETs prevent the loss of cav-1, resulting in increased mitochondrial targeting and cardioprotection.

Matrix metalloproteinases (MMPs) are known to degrade components of the extracellular matrix. More recently, in myocardial oxidative stress injury including ischemia-reperfusion, MMP-2 is activated and degrades troponin I and α-actinin. MMP activity is regulated at several levels. We recently showed that MMP-2 is localized in the caveolae of cardiomyocytes and is negatively regulated by caveolin-1 (Cav-1). The caveolin scaffolding domain of Cav-1 inhibits MMP-2 proteolytic activity in vitro, and Cav-1(-/-) mouse hearts have increased MMP-2 activity compared with controls. Whether this increase in MMP-2 activity translates to impaired cardiac function is unknown. Hearts isolated from Cav-1(-/-) mice and their wild-type controls were perfused as isolated working hearts and physiologically challenged with increasing increments of left atrial preload (7-22.5 mmHg). The hearts were then pharmacologically challenged with increasing concentrations of isoproterenol (0.1-100 nM). Functionally, the Cav-1(-/-) hearts were similar to the controls in heart rate, peak systolic pressure, developed pressure, and rate pressure product. At higher preload pressures, the Cav-1(-/-) hearts outperformed the control hearts. Coronary flow was significantly higher in Cav-1(-/-) hearts under all conditions. The highest concentration of isoproternol increased the heart rate of Cav-1(-/-) hearts more than in controls. Western blot analysis revealed no significant changes in troponin I or α-actinin between Cav-1(-/-) hearts and their controls. There was a significant loss of MMP-2 from both knockout and control hearts during the perfusion. In summary, despite the loss of Cav-1, Cav-1(-/-) hearts show similar or better cardiac function compared with wild-type hearts following physiological challenge or β-adrenergic stimulation in vitro, and this appears unrelated to changes in MMP-2.

Persistent arterial hypotension is a hallmark of sepsis and is believed to be caused, at least in part, by excess nitric oxide (NO). NO can combine with superoxide to produce peroxynitrite, which activates matrix metalloproteinases (MMPs). Whether MMP inhibition in vivo protects against vascular hyporeactivity induced by endotoxemia is unknown. Male Sprague-Dawley rats were administered either bacterial lipopolysaccharide (LPS, 4 mg/kg ip) or vehicle (pyrogen-free water). Later (30 min), animals received the MMP inhibitor doxycycline (4 mg/kg ip) or vehicle (pyrogen-free water). After LPS injection (6 h), animals were killed, and aortas were excised. Aortic rings were mounted in organ baths, and contractile responses to phenylephrine or KCl were measured. Aortas and plasma were examined for MMP activity by gelatin zymography. Aortic MMP and inducible nitric oxide synthase (iNOS) were examined by immunoblot and/or immunohistochemistry. Doxycycline prevented the LPS-induced development of ex vivo vascular hyporeactivity to phenylephrine and KCl. iNOS protein was significantly upregulated in aortic homogenates from endotoxemic rats; doxycycline did not alter its level. MMP-9 activity was undetectable in aortic homogenates from LPS-treated rats but significantly upregulated in the plasma; this was attenuated by doxycycline. Plasma MMP-2 activities were unchanged by LPS. Specific MMP-2 activity was increased in aortas from LPS-treated rats. This study demonstrates the in vivo protective effect of the MMP inhibitor doxycycline against the development of vascular hyporeactivity in endotoxemic rats.

Matrix metalloproteinase‐2 (MMP‐2) proteolyzes troponin I (TnI), myosin light chain‐1 (MLC‐1) and α‐actinin within cardiomyocytes in hearts challenged with ischemia and reperfusion (I/R).MMP‐2 is also co‐localized with caveolin‐1 (Cav‐1) in cardiomyocytes. Cav‐1 inhibits MMP‐2 activity. Cav‐1 null (Cav‐1−/−) hearts have increased MMP‐2 activity. How this affects heart function in response to physiological and pharmacological challenges, and whether this has an impact on known MMP‐2 substrates, is unknown.Cav‐1−/− or control (Cav‐1+/+) hearts were isolated and perfused as working hearts. Hearts were perfused aerobically for 30 min followed by both physiological (preload varied between 7‐22.5 mmHg) and pharmacological (0.1‐100 nM isoproterenol) challenges.Cardiac function between the Cav‐1−/− vs Cav‐1+/+ hearts did not differ in either challenge. Gelatin zymography of heart homogenates showed no differences in MMP‐2 activity. Protein levels of MMP‐2, TnI, MLC‐1 and α‐actinin were similar between Cav‐1+/+ and Cav‐1−/− hearts. Hearts briefly perfused for 10 min showed more MMP‐2 activity compared with hearts perfused for 90 min. Prolonged perfusion and the oxidative stress inherent in the isolated working heart procedure may release MMP‐2 from the heart. MMP‐2 proteolysis of intracellular proteins may be dependent upon its activation in I/R.This work is supported by HSF, CIHR and AHFMR.

Some receptors and signaling molecules, such as Rho-kinase (ROCK), localize in caveolae. We asked whether the function of histamine receptors (H(1)) and 5-hydroxytryptamine (serotonin) receptors (5-HT(2A)) in bovine tracheal smooth muscle are modified after caveolae disruption and if so, whether the altered ROCK activity plays a role in this modification. Methyl-beta-cyclodextrin (MbetaCD), used to deplete membrane cholesterol, was shown to disrupt caveolae and diminish sustained contractions to histamine (approximately 80%), 5-HT (100%), alpha-methyl-5-HT (100%), and KCl (approximately 30%). Cholesterol-loaded MbetaCD (CL-MbetaCD) restored the responses to KCl and partially restored the responses to agonists. ROCK inhibition by Y-27632 diminished contractions to histamine (approximately 85%) and 5-HT (approximately 59%). 5-HT or histamine stimulation augmented ROCK activity. These increases were reduced by MbetaCD and partially reestablished by CL-MbetaCD. The increase in intracellular Ca(2+) that was induced by both agonists was reduced by MbetaCD. The presence of caveolin-1 (Cav-1), H1, 5-HT(2A), and ROCK1 was corroborated by immunoblotting of membrane fractions from sucrose gradients and by confocal microscopy. H(1) receptors coimmunoprecipitated with Cav-1 in caveolar and noncaveolar membrane fractions, whereas 5-HT(2A) receptors appeared to be restricted to noncaveolar membrane fractions. We conclude that caveolar and cholesterol integrity are indispensable for the proper functionality of the H(1) and 5-HT(2A) receptors through their Rho/ROCK signaling.

Neuronal nitric oxide synthase (nNOS) in myenteric neurons is activated during peristalsis to produce nitric oxide which relaxes intestinal smooth muscle. A putative nNOS is also found in the membrane of intestinal smooth muscle cells in mouse and dog. In this study we studied the possible functions of this nNOS expressed in mouse small intestinal smooth muscle colocalized with caveolin-1(Cav-1). Cav-1 knockout mice lacked nNOS in smooth muscle and provided control tissues. 60 mM KCl was used to increase intracellular [Ca(2+)] through L-type Ca(2+) channel opening and stimulate smooth muscle NOS activity in intestinal tissue segments. An additional contractile response to LNNA (100 microM, NOS inhibitor) was observed in KCl-contracted tissues from control mice and was almost absent in tissues from Cav-1 knockout mice. Disruption of caveolae with 40 mM methyl-beta cyclodextrin in tissues from control mice led to the loss of Cav-1 and nNOS immunoreactivity from smooth muscle as shown by immunohistochemistry and a reduction in the response of these tissues to N-omega-nitro-L-arginine (LNNA). Reconstitution of membrane cholesterol using water soluble cholesterol in the depleted segments restored the immunoreactivity and the response to LNNA added after KCl. Nicardipine (1 microM) blocked the responses to KCl and LNNA confirming the role of L-type Ca(2+) channels. ODQ (1 microM, soluble guanylate cyclase inhibitor) had the same effect as inhibition of NOS following KCl. We conclude that the activation of nNOS, localized in smooth muscle caveolae, by calcium entering through L-type calcium channels triggers nitric oxide production which modulates muscle contraction by a cGMP-dependent mechanism.

Plasma membrane calcium ATPase (PMCA) is an important calcium extrusion mechanism in smooth muscle cells with PMCA4 as the predominant isoform. PMCA is localized in lipid rafts and caveolae. In this study we examined the effects of blocking PMCA4 function in small intestinal tissue from wild type (Cav1+/+) and caveolin‐1 knockout (Cav1−/−) mice and in bovine airway smooth muscle tissue before and after caveolae disruption. Small intestinal tissues from Cav1+/+ mice treated with the PMCA4 inhibitor caloxin 1c2 (5 μM) showed a higher contractile tone to carbachol (10 μM) when compared to tissues treated with 5 μM of a control scrambled peptide. This effect of caloxin 1c2 was not seen in tissues from Cav1−/− mice. Immunohistochemistry and Western blotting showed that PMCA was co‐localized with caveolin‐1 in Cav1+/+ tissues. In bovine tracheal smooth muscle tissue, caveolae disruption by cholesterol depletion led to the diminution of caveolin‐1 and PMCA4b immunoreactivities, previously co‐localized in the smooth muscle plasma membrane, and to the loss of the increase in carbachol‐induced contraction by caloxin 1c2. Our results suggest that the calcium removal function of PMCA4 in smooth muscle cells is dependent on its presence in intact caveolae. We suggest that this is due to the close spatial arrangement that allows calcium extrusion from a privileged cytosolic space between caveolae and sarcoplasmic reticulum.

Matrix metalloproteinase-2 (MMP-2) may play roles at intracellular and extracellular sites of the heart in ischaemia/reperfusion injury. Caveolins (Cav-1, -2 and -3) are lipid raft proteins which play roles in cell signalling. This study examined, using immunohistochemistry and two photon confocal microscopy, if MMP-2 and caveolins co-localize at the plasma membrane of cardiac cells: cardiomyocytes (CM), fibroblasts (FB) and capillary endothelial cells (CEC) in the left ventricle (LV) of the Cav-1(+/+) and Cav-1(-/-) mouse heart. In Cav1(+/+) mouse LV MMP-2 and Cav-1 co-localized at CM plasma membranes, and at multiple locations in FB and CEC. MMP-2 co-localized with Cav-2 only at CEC. MMP-2 co-localized with Cav-3 at CM plasma membranes and Z-lines, and partially at FB and CEC. In Cav-1-/-LV Cav-1 and MMP-2 were absent or reduced everywhere. Cav-2 appeared at CEC despite the absence of Cav-1. Cav-3 appeared at CM plasma membranes and Z-lines, FB and CEC. Also, FAK in FB and c-Kit in interstitial Cajal-like cells (ICLC) were completely absent. By transmission electron microscopy in Cav-1(+/+), regular size caveolae (Cav) were at CEC, irregular size Cav were at CM and a few were at FB. In Cav-1(-/-) there were few Cav at CM and FB and some at CEC. To conclude, MMP-2 is closely associated with caveolins at FB and CEC as well as at CM. Also, MMP-2 is closely associated with FAK at FB and c-Kit at ICLC. Thus, Cav-1 expression is not necessary for Cav-2 expression. Cav-3 or Cav-3 with Cav-2 has the capability to make Cav.

Neuronal nitric oxide synthase (nNOS) expressed in muscle cells modulates contraction in skeletal and cardiac muscle. In this study, we examine the possible function of nNOS in mouse small intestinal smooth muscle. We examined the tissue response to LNNA (NOS inhibitor) following activation of L‐type calcium channels by depolarization of intact small intestinal segments by KCl. Tissues from control mice responded with an increase in the contractile tone. However, tissues from caveolin‐1 knockout mice, which lacked the nNOS expressed in intestinal smooth muscle upon immunohistochemical examination, did not show a response to LNNA. Immunoprecipiation of caveolin‐1 pulled down nNOS indicating their interaction in smooth muscle membrane. Further, caveolae disruption by cholesterol depletion in control mice led to the loss of caveolin‐1 and nNOS immunoreactivity form smooth muscle membrane and the loss of the tissue response to LNNA. On the other hand, repletion of membrane cholesterol restored nNOS and caveolin‐1 immunoreactivity together with the increase in contractile tone following the treatment with LNNA. The involvement of L‐type calcium channels was examined using nicardipine which blocked the responses to KCl and LNNA. Increasing the intracellular calcium with a SERCA pump inhibitor did not have the same effect as KCl. Blocking soluble guanylate cyclase had the same effect of NOS inhibition while potassium channel blockers did not. We concluded that the activation of nNOS, localized in smooth muscle caveolae, by calcium entering through L‐type calcium channels triggers NO production which modulates the contraction by activation of soluble guanylate cyclase.

beta-Adrenoceptors are G protein-coupled receptors whose functions are closely associated with caveolae in the heart and cultured cell lines. In the gut, they are responsible, at least in part, for the mediation of the sympathetic stimulation that might lead to intestinal paralysis postoperatively. We examined the effect of caveolin-1 knockout on the beta-adrenoceptor response in mouse small intestine. The relaxation response to (-)-isoprenaline in carbachol-contracted small intestinal tissue segments was reduced in caveolin-1 knockout mice (cav1(-/-)) compared with their genetic controls (cav1(+/+)). Immunohistochemical staining showed that beta-adrenoceptor expression was similar in both strains in gut smooth muscle. Selective beta-adrenoceptor blockers shifted the concentration response curve (CRC) of (-)-isoprenaline to the right in cav1(+/+) intestine, but not in cav1(-/-), with greatest shift in case of the beta(3)-blocker, SR59230A. The CRC of the selective beta(3)-agonist BRL 37344 was also shifted to the right in cav1(-/-) compared with cav1(+/+). The cAMP-dependent protein kinase (PKA) inhibitor H-89 shifted the CRC of (-)-isoprenaline to the right in cav1(+/+) but not in cav1(-/-). H-89 reduced the relaxation due to forskolin and dibutyryl cAMP in cav1(+/+) but not in cav1(-/-), suggesting a reduction in PKA activity in cav1(-/-). In cav1(+/+), PKA was colocalized with caveolin-1 in the cell membrane, but PKA immunoreactivity persisted in cav1(-/-). Examination of PKA expression in the lipid raft-rich membrane fraction of the jejunum revealed reduced PKA expression in cav1(-/-) compared with cav1(+/+). The results of the present study show that the function of beta-adrenoceptors is reduced in cav1(-/-) small intestine likely owing to reduced PKA activity.

Pacing of intestinal smooth muscle is driven by a network of cells found in the myenteric plexus called the interstitial cells of Cajal (ICC-MP), which produce a rhythmic pacemaker current. Using i...

Confocal microscopic images were obtained from the immunohistochemical sections of jejeunum to determine the localization/colocalization between caveolin-1, caveolin-2 and caveolin-3 in intestinal smooth muscle cells (SMCs) and interstitial cells of Cajal (ICC) of Cav1+/+ and Cav1−/− mouse. Intestinal regions were segmented [inner circular muscle (icm), outer circular muscle (ocm), myenteric plexus region (mp), and longitudinal muscle (lm)] by LSM 5 and analyzed by ImageJ to show Pearson’s correlation (r p) and overlap coefficient (r) of colocalization. In the intestine of Cav1+/+, caveolin-1 (cav1) was colocalized with caveolin-2 (cav2) and caveolin-3 (cav3). Cav2 also was well colocalized with cav3. In the intestine of Cav1−/−, cav1 and cav2 were absent in all images, but reduced cav3 was expressed in ocm. Caveolae were present in cell types with cav1 in Cav1+/+, and present with cav3 in ocm of Cav1−/−. C-kit occurred in deep muscular plexus (ICC-DMP) and myenteric plexus (ICC-MP), in both Cav1+/+ and Cav1−/−, and colocalized with cav1 and cav2 in the intestine of Cav1+/+. Cav3 was absent/present at low immunoreactivity in ICC-DMP and ICC-MP of the intestines of Cav1+/+ and Cav1−/−. To conclude, cav1 is necessary for the expression of cav2 in SMC and ICC of intestine and facilitates, but is not necessary for the expression of cav3.

Glucagon-like polypeptide 1 (GLP-1) may be amajor enterogastrone, slowing gastric emptying whenreleased by intestinal nutrients. In six conscious dogs,we studied the effects of GLP-1, on antropyloric motility, gastric emptying, and transpyloricflow after instillation of 500 ml of saline into thestomach. The meal was given and recordings were started15 min after intravenous bolus and infusion of either saline or three different doses of GLP-1.Intravenous GLP-1 produced a dose-related retardation ofgastric emptying associated with a decrease in thenumber and volume of flow pulses in comparison tosaline. This change in transpyloric flow was associatedwith an inhibition of antropyloric pressure waves, astimulation of isolated pyloric pressure waves, and anincrease in basal pyloric tone induced by intravenous GLP-1 infusion. Our findings show that GLP-1has a potent dose-dependent inhibitory effect ontranspyloric flow and gastric emptying. This effect istemporally associated with inhibition of antral“pumping” and stimulation of pyloric“braking” mechanisms.

The role of nitric oxide (NO) mechanisms incontrol of pyloric function and transpyloric flow wereinvestigated in six conscious dogs. Antropyloroduodenalmotility, transpyloric flow, and gastric emptying were measured 15 min after intravenousinjection of 100 ml of either saline, L-arginine (50mg/kg), L-NNA (5 mg/kg), or L-arginine (50 mg/kg)followed by L-NNA (5 mg/kg). Infusion of L-NNA wasassociated with retardation of gastric emptying (65± 6%) in the first 30 min, in comparison to thesaline (90 ± 3%) or L-arginine (90 ± 2%).This effect was prevented by infusion of L-arginineprior to L-NNA, after which 89 ± 3% of the liquidemptied in 30 min. There was a significant reduction (P< 0.05) in the number and volume of flow pulses, andan increase in pyloric tone (P < 0.05) after L-NNA in comparison to the other three testconditions. There were no differences, however, in thenumber of antropyloric or isolated pyloric pressurewaves under the four conditions. Our findings suggestthat NO mechanisms influence gastric emptying andtranspyloric flow of nonnutrient liquids by altering thepyloric tone, thus increasing resistance toflow.

Ca2+ plays an important role in muscle contraction. Two major Ca2+ release channels have been suggested to be present in smooth muscle by functional studies: one is sensitive to inositol 1,4,5-trisphosphate (IP3) and the other is operated by Ca2+ and also sensitive to ryanodine. The existence of these two channels in smooth muscle has been confirmed recently by radioligand binding studies using subcellular membrane and named IP3 and ryanodine receptors. The isolated IP3 and ryanodine receptors from smooth muscles show a channel activity and can be operated by IP3 and ryanodine, respectively. Some similarities of these two receptors have been noted at the molecular level, but they are most likely to be two separate proteins. Although both channels are believed to be involved in regulating cytosol Ca2+ level in smooth muscle, their relative importance in different smooth muscles is still not very clear.