Reperfusion and calculated RISKs: pharmacological postconditioning of human myocardium.

The last five years have witnessed a remarkable resurgence of interest in myocardial reperfusion injury. Reperfusion is absolutely essential to salvage ischaemic myocardium but experimental and clinical studies show that reperfusion-associated injury may mask the full benefits of prompt reperfusion in acute myocardial infarction. In the current issue of the British Journal of Pharmacology, Mudalagiri et al demonstrate a protective effect against simulated reperfusion injury using exogenously applied erythropoietin in human isolated myocardium. Crucially, the benefits of erythropoietin were observed when it was administered specifically during re-oxygenation. The demonstration that the protective effects of the cytokine were dependent on PI3-kinase/Akt and ERK1/2 activation provides compelling evidence that reperfusion injury salvage kinases (RISKs) are key survival mechanisms in human myocardium, as they are in experimental animal species. Although erythropoietin may be only one of several potential pharmacological approaches in human patients, this study establishes the important proof-of-principle that activation of RISKs is protective in human myocardium and could be a promising therapeutic target in acute myocardial infarction.

Cyclic GMP and protein kinase-G in myocardial ischaemia-reperfusion: opportunities and obstacles for survival signaling.

It is clear that multiple signalling pathways regulate the critical balance between cell death and survival in myocardial ischaemia-reperfusion. Recent attention has focused on the activation of survival or salvage kinases, particularly during reperfusion, as a common mechanism of many cardioprotective interventions. The phosphatidyl inositol 3'-hydroxy kinase/Akt complex (PI3K/Akt) and p42/p44 mitogen-activated protein kinase cascades have been widely promoted in this respect but the cyclic guanosine 3',5'-monophosphate/cGMP-dependent protein kinase (cGMP/PKG) signal transduction cassette has been less systematically investigated as a survival cascade. We propose that activation of the cGMP/PKG signalling pathway, following activation of soluble or particulate guanylate cyclases, may play a pivotal role in survival signalling in ischaemia-reperfusion, especially in the classical preconditioning, delayed preconditioning and postconditioning paradigms. The resurgence of interest in reperfusion injury, largely as a result of postconditioning-related research, has confirmed that the cGMP/PKG pathway is a pivotal salvage mechanism in reperfusion. Numerous studies suggest that the infarct-limiting effects of preconditioning and postconditioning, exogenously donated nitric oxide (NO), natriuretic peptides, phosphodiesterase inhibitors, and other diverse drugs and mediators such as HMG co-A reductase inhibitors (statins), Rho-kinase inhibitors and adrenomedullin, whether given before and during ischaemia, or specifically at the onset of reperfusion, may be mediated by activation or enhancement of the cGMP pathway, either directly or indirectly via endogenous NO generation downstream of PI3K/Akt. Putative mechanisms of protection include PKG regulation of Ca(2+) homeostasis through the modification of sarcoplasmic reticulum Ca(2+) uptake mechanisms, and PKG-induced opening of ATP-sensitive K(+) channels during ischaemia and/or reperfusion. At present, significant technical obstacles in defining the precise roles played by cGMP/PKG signalling include the heavy reliance on pharmacological PKG inhibitors of uncertain selectivity, difficulties in determining PKG activity in intact tissue, and the growing recognition that intracellular compartmentalisation of the cGMP pool may contribute markedly to the nucleotide's biological actions and biochemical determination. Overall, the body of experimental evidence suggests that cGMP/PKG survival signalling ameliorates irreversible injury associated with ischaemia-reperfusion and may be a tractable therapeutic target.

Glimepiride, a novel sulfonylurea, does not abolish myocardial protection afforded by either ischemic preconditioning or diazoxide.

BACKGROUND: The sulfonylurea glibenclamide (Glib) abolishes the cardioprotective effect of ischemic preconditioning (IP), presumably by inhibiting mitochondrial K(ATP) channel opening in myocytes. Glimepiride (Glim) is a new sulfonylurea reported to affect nonpancreatic K(ATP) channels less than does Glib. We examined the effects of Glim on IP and on the protection afforded by diazoxide (Diaz), an opener of mitochondrial K(ATP) channels. METHODS AND RESULTS: Rat hearts were Langendorff-perfused, subjected to 35 minutes of regional ischemia and 120 minutes of reperfusion, and assigned to 1 of the following treatment groups: (1) control; (2) IP of 2x 5 minutes each of global ischemia before lethal ischemia; or pretreatment with (3) 30 micromol/L Diaz, (4) 10 micromol/L Glim, (5) 10 micromol/L Glib, (6) IP+Glim, (7) IP+Glib, (8) Diaz+Glim, or (9) Diaz+Glib. IP limited infarct size (18.5+/-1% vs 43.7+/-3% in control, P<0.01) as did Diaz (22.2+/-4.7%, P<0.01). The protective actions of IP or Diaz were not abolished by Glim (18.5+/-3% in IP+Glim, 22.3+/-3% in Diaz+Glim; P<0.01 vs control). However, Glib abolished the infarct-limiting effects of IP and Diaz. Patch-clamp studies in isolated rat ventricular myocytes confirmed that both Glim and Glib (each at 1 micromol/L) blocked sarcolemmal K(ATP) currents. However, in isolated cardiac mitochondria, Glim (10 micromol/L) failed to block the effects of K(ATP) opening by GTP, in contrast to the blockade caused by Glib. CONCLUSIONS: Although it blocks sarcolemmal currents in rat cardiac myocytes, Glim does not block the beneficial effects of mitochondrial K(ATP) channel opening in the isolated rat heart. These data may have significant implications for the treatment of type 2 diabetes in patients with ongoing ischemic heart disease.

Adaptation to myocardial stress in disease states: is preconditioning a healthy heart phenomenon?

Effective therapeutic strategies for protecting the ischaemic myocardium are much sought after. Ischaemic heart disease in humans is a complex disorder, often associated with other systemic diseases such as dyslipidaemia, hypertension and diabetes that exert multiple biochemical effects on the heart, independently of ischaemia. Ischaemic preconditioning of myocardium is a well-described adaptive response in which brief exposure to ischaemia markedly enhances the ability of the heart to withstand a subsequent ischaemic insult. The underlying molecular mechanisms of this phenomenon have been extensively investigated in the hope of identifying new rational approaches to therapeutic protection of the ischaemic myocardium. However, most studies have been undertaken in animal models in which ischaemia is imposed in the absence of other disease processes. In this article, Peter Ferdinandy, Zoltan Szilvassy and Gary Baxter review the ways in which systemic diseases might modify the preconditioning response and they emphasize the importance of further preclinical studies that specifically examine preconditioning in relation to complicating disease states.

Prolonging the delayed phase of myocardial protection: repetitive adenosine A1 receptor activation maintains rabbit myocardium in a preconditioned state.

OBJECTIVES: This study was designed to examine whether the myocardium can be maintained in a protected state by extending the delayed phase of cardioprotection with chronic, intermittent adenosine A1 receptor activation. BACKGROUND: Several recent studies have explored the temporal characteristics of the protective effects of ischemic preconditioning. Two distinct phases of myocardial protection have been described: the short-lived immediate phase, or "classic" preconditioning, and the delayed phase, or "second window of protection" (SWOP). Previous studies have examined the potential for extending the duration of classic preconditioning by repeated application of the preconditioning stimulus. Pretreatment with either multiple episodes of ischemia or continuous infusion of a selective adenosine A1 receptor agonist, 2-chloro-N6-cyclopentyladenosine (CCPA), resulted in attenuation of the protective effects of preconditioning, implying downregulation of the receptors involved in triggering classic preconditioning. METHODS: Male New Zealand White rabbits were treated with repeated intravenous boluses of CCPA, 100 microg/kg body weight, or 0.9% saline at 48-h intervals. Forty-eight hours after the fifth dose (day 10), the animals were anesthetized and subjected to 30 min of coronary occlusion, followed by 120 min of reperfusion. Infarct size was determined as a percentage of myocardial risk volume using tetrazolium staining. To further explore whether the rabbits had developed tolerance to the effects of adenosine A1 receptor activation, a subgroup of animals were treated with a further bolus of CCPA, 100 microg/kg, at the end of the reperfusion period, and the hemodynamic response was monitored for 10 min before excision of the heart. RESULTS: Pretreatment with intermittent doses of CCPA resulted in a 42% reduction in the infarct to risk ratio compared with vehicle pretreatment (26.6+/-3.7% vs. 45.9+/-5.5%, p < 0.01). Furthermore, CCPA treatment at the end of reperfusion resulted in identical hypotension and bradycardia in both groups. CONCLUSIONS: We conclude that rabbits can be maintained in a protected state against myocardial infarction by repeated activation of adenosine A1 receptors, with no evidence of tachyphylaxis to the infarct-limiting or hemodynamic effects of CCPA. This finding suggests that adenosine A1 receptor activation may hold promise as a new approach to long-term cardioprotection.

Angiotensin-converting enzyme inhibition enhances a subthreshold stimulus to elicit delayed preconditioning in pig myocardium.

OBJECTIVES: We assessed the effect of angiotensin-converting enzyme (ACE) inhibition in combination with a subthreshold preconditioning (PC) stimulus to elicit delayed preconditioning against infarction in pig myocardium. BACKGROUND: Bradykinin triggers early PC. Angiotensin-converting enzyme inhibitors increase local bradykinin levels via inhibition of kinin breakdown and have been shown in experimental studies to augment early protection afforded by PC. A role for bradykinin in eliciting delayed PC has not so far been identified. METHODS: We used a two-day protocol. On day 1 (closed chest), pigs were either sham-operated (group 1) or preconditioned, using balloon catheter inflation of the left anterior descending (LAD) coronary artery, with either a full (4 x 5 min PC, group 2) or subthreshold PC stimulus (2 x 2 min PC, group 3). Additional groups were pre-treated with perindoprilat (0.06 mg/kg i.v.) before sham (group 4) or subthreshold PC (group 5). On day 2 (open chest), all pigs were subjected to 40 min occlusion of the LAD followed by 3 h of reperfusion. Infarct size was determined by tetrazolium staining. RESULTS: Group 1 had a mean infarct size of 42.8+/-3.2% of the risk zone. Preconditioning with 4 x 5 min reduced the infarct size to 19.5+/-3.9% (p < 0.05). Groups 3 and 4 had infarct sizes not statistically different from group 1. However, combining perindoprilat with subthreshold PC resulted in a significant limitation of the infarction (18.4+/-3.1% p < 0.05), comparable with group 2. CONCLUSIONS: This is the first study to show that ACE inhibition can augment a mild ischemic stimulus to induce a protected state 24 h later.

Protection of the myocardium by ischaemic preconditioning: mechanisms and therapeutic implications.

Preconditioning of the heart with brief periods of ischaemia protects the myocardium for up to 90 min against a more sustained ischaemic injury. A "second window of protection" occurs 24 hr after preconditioning with ischaemia. The cardioprotective effects of ischaemic preconditioning involve the release of mediators (adenosine, bradykinin, catecholamines, prostaglandins, endothelin-1), which either alone or in concert activate protein kinase C, which translocates to the cell membrane. This manuscript reviews (i) the cardioprotective effects of ischaemic preconditioning, (ii) the underlying mechanisms, (iii) the effects of ischaemic preconditioning of other tissues (skeletal muscle, brain and kidney), and (iv) the clinical implications.

Representational difference analysis of cDNA identifies novel genes expressed following preconditioning of the heart.

Preconditioning of the myocardium rapidly induces a number of transcription factors, which are likely to be responsible for a cascade of transcriptional changes underlying the development of delayed adaptation. Identifying these changes provides insight into the molecular pathways elicited by sub-lethal ischaemia and the mechanism leading to delayed adaptation. Genes up-regulated in rabbit myocardium in vivo by ischaemic preconditioning following reperfusion for 2 h, 4 h and 6 h post-treatment were identified by representational difference analysis of cDNA (cDNA. RDA). The area of the left ventricle rendered ischaemic by preconditioning or the equivalent area of sham-treated animals was isolated and cDNA.RDA performed. Three novel genes and six genes with known function where identified, including the TGFbeta receptor interacting protein 1, the alpha isoform of the A subunit of PP2 and the cap binding protein NCBP1. To determine whether expression of these genes correlated with preconditioning per se, expression was measured in myocardium after both ischaemic as well as heat shock induced preconditioning following 2 h, 4 h, and 6 h reperfusion. These genes were induced in rabbit myocardium in vivo by both ischaemia and heat shock, consistent with a fundamental role in the development of delayed adaptation. The well described role of PP2 in modulating the mitogen-activated protein kinase pathway and promoting cell survival is consistent with our previous work, which identified the reperfusion injury salvage kinase pathway in mediating the protective effects of ischaemic preconditioning. Expression of Trip1 and Ncbp1 also implicates TGFbeta signalling pathways and RNA processing and transport in delayed adaptation to stress in the myocardium.

Reperfusion injury revisited: is there a role for growth factor signaling in limiting lethal reperfusion injury?

Myocardial reperfusion injury represents an important therapeutic target. The ability of several peptide growth factors, including transforming growth factor-beta1, insulin, insulin-like growth factor-1, cardiotrophin-1 and fibroblast growth factors, to modify reperfusion injury has been examined in recent studies. The protective effects of these agents may be related to the inhibition of apoptosis, especially during reperfusion, probably through p42/p44 MAP kinase and PI3-kinase/Akt signaling. Growth factor signaling may therefore represent a novel approach for the development of pharmacological strategies that attenuate reperfusion injury in the heart.

Role of adenosine in delayed preconditioning of myocardium.

Myocardial protection conferred by ischemic preconditioning occurs in a bimodal time course. The early cardioprotection wanes rapidly and is succeeded by a delayed phase of protection reducing infarct development, myocardial stunning and arrhythmias. This 'second window' of preconditioning may be evident for up to 72 h. The current mechanistic paradigm for delayed preconditioning against infarction invokes roles for several freely-diffusible molecules, generated during the preconditioning period, that act in autocrine and/or paracrine fashion as triggers of cellular adaptation. These include adenosine, nitric oxide, reactive oxygen species and bradykinin. A role for adenosine receptor activation as a proximal molecular mechanism leading to delayed preconditioning against infarction was established in 1994. Pharmacological adenosine receptor blockade during preconditioning abolishes the acquisition of delayed protection, while transient adenosine A(1) or A(3) receptor activation fully recapitulates protection against infarction (but not against stunning or arrhythmias) 24 h later. Although nitric oxide is a co-trigger of delayed preconditioning, A(1) agonist-induced delayed protection is independent of nitric oxide production. Adenosine receptor activation causes the activation of a complex protein kinase signalling cascade and, putatively, the subsequent activation of gene transcription. The induction or post-translational regulation of several proteins is associated with A(1) agonist-induced delayed protection. These include the mitochondrial manganese-conjugated superoxide dismutase, and the 27-kDa heat shock protein. Opening of K(ATP) channels during the index ischaemic event is an obligatory downstream event mediating A(1) and A(3) agonist induced delayed protection. However, the mechanism of sub-acute regulation of K(ATP) channels following adenosine receptor activation is unknown. Evidence for induction of inducible nitric oxide synthase as a distal mechanism of A(1) agonist-induced delayed protection is equivocal.

Ischaemic preconditioning protects hypertrophied myocardium.

OBJECTIVE: Clinical and experimental evidence suggests that hypertrophied myocardium is more susceptible to injury from ischaemia than normal myocardium. This study was designed to investigate whether preconditioning confers protection on hearts with moderate hypertrophy. METHODS: Cardiac hypertrophy induced by hypertension was produced in rats by giving saline drinking fluid and subcutaneous deoxycorticosterone acetate for four weeks. After thoracotomy, localisation of the left main coronary artery and stabilisation, groups of hypertensive animals with cardiac hypertrophy (HT) and their age matched normotensive controls (NT) underwent a preconditioning protocol consisting of 5 min occlusion of the left main coronary artery followed by 10 min reperfusion. This was followed by 45 min ischaemia and at least 2.5 h reperfusion. Control animals in the hypertrophied and normotensive groups were treated identically but were not subjected to the preconditioning protocol. Thus there were four experimental groups: HT preconditioned (n = 8), HT control (n = 7), NT preconditioned (n = 7), and NT control (n = 7). Infarct size was measured using triphenyl tetrazolium chloride and was expressed as a percentage of the volume at risk, measured with fluorescent particles. RESULTS: Heart weight was greater (p < 0.01) in the HT groups than in the NT groups [HT preconditioned 1.7(SEM 0.06) g, HT control 1.71(0.02) g, NT preconditioned 1.3(0.04) g, NT control 1.31(0.03) g]. Infarct to risk volume ratio (I/R) was significantly lower (p < 0.05) in the preconditioned groups as compared with controls [HT preconditioned 19.1(1.5)% v HT control 67.1(5.6)%, and NT preconditioned 33.4(5.5)% v NT control 77.1(3.8)%]. There was no significant difference in the volume at risk of infarction between the four groups. CONCLUSIONS: Ischaemic preconditioning can induce myocardial protection in hypertrophied myocardium; this is the first study to demonstrate preconditioning in hypertrophy.

Ischaemic preconditioning: present position and future directions.

Preconditioning the myocardium using short episodes of sublethal ischaemia will delay the onset of necrosis during a subsequent lethal ischaemic insult. This powerful protective adaptation of the myocyte has also been observed in other cell types. The potential for clinical application to benefit patients with a variety of pathological conditions has led to an expansion in our knowledge concerning the pathophysiology of ischemia-reperfusion injury and the regulatory mechanisms underlying cellular metabolism. We feel it is timely to assess the current position in this field and provide a critical appraisal to facilitate future research.

Infarct limitation of the second window of protection in a conscious rabbit model.

OBJECTIVES: Myocardial protection associated with ischemic preconditioning (PC) wanes within an hour or two. It has recently been observed, however, that a delayed phase of protection appears about 24 h after ischemic PC in anesthetized rabbits and dogs which might be related to synthesis of cytoprotective proteins. We tested whether a second window of protection could be induced in conscious rabbits. METHODS: Rabbits chronically instrumented with a coronary artery occluder and ECG electrodes experienced a 30-min coronary occlusion followed by 3 h reperfusion. Infarct size was measured with triphenyltetrazolium chloride. RESULTS: 35.7 +/- 2.3% of the risk zone infarcted in control animals. PC with 4 cycles of 5-min coronary occlusion/10-min reperfusion 24 h prior to the 30-min ischemia decreased infarction to 24.1 +/- 1.4% of the risk zone (P < 0.01). During the 30-min occlusion 3 of 7 non-PC rabbits developed ventricular fibrillation, while this arrhythmia did not occur in the 7 PC animals (P < 0.1). Myocardial hsp70 content in PC rabbits was twice that in controls. Collateral blood flow was not different in the two groups. CONCLUSIONS: A second window of protection exists in conscious rabbits which minimizes both infarction and arrhythmias, and cytoprotective protein content is increased in the myocardium of protected animals.

Quantitative assessment of cardiac myocyte apoptosis in tissue sections using the fluorescence-based tunel technique enhanced with counterstains.

Apoptosis is a distinct form of cell death, induced, for example, by ischaemia/reperfusion injury, that results in characteristic alterations in cell morphology and fate. In tissue sections, the most commonly used technique to detect apoptosis is terminal deoxynucleotidyl transferase mediated nick end labelling (TUNEL) staining which labels the ends of DNA strand breaks characteristic of the apoptotic process. However, without the employment of additional staining, TUNEL is only a qualitative procedure that gives no information about the proportion of negative cells nor the cell type undergoing apoptosis. We have utilised propidium iodide (PI) as a counterstain to visualise TUNEL negative nuclei together with anti-desmin antibody in order to assess quantitatively apoptosis in specific cell types. The procedure has been evaluated in tissue sections from isolated perfused rat hearts subjected to ischaemia and reperfusion. Hearts were cross-sectioned into four 2.5 mm thick slices which were fixed in 4% formaldehyde and embedded in paraffin. Serial sections (5 microns) were cut, dewaxed and pretreated by incubation with trypsin at 37 degrees C for 30 min. After the employment of the TUNEL assay, sections were labelled with anti-desmin antibody, counterstained with PI and finally examined by confocal fluorescent microscopy. Apoptosis was not seen in sections from hearts subjected to ischaemia alone nor in control hearts. After 35 min of ischaemia the percentages of TUNEL positive cells were very low both in myocytes (0.1%) and in non-myocytes (0.3%). In ischaemic-reperfused hearts, the number of TUNEL positive cells was only significantly higher in vascular cells (44+/-5%) and cardiac myocytes (6+/-2%). This simple method therefore allows quantification of apoptosis in myocytic and non-myocytic cells in tissue sections. Use of alternative immunohistochemical markers would permit adaptation of the method to the quantitative assessment of apoptosis in other tissues.

Pharmacological evidence that inducible nitric oxide synthase is a mediator of delayed preconditioning.

Brief periods of myocardial ischaemia preceding a subsequent more prolonged ischaemic period 24-72 h later confer protection against myocardial infarction ('delayed preconditioning' or the 'second window' of preconditioning). In the present study, we examined the effects of pharmacological modifiers of inducible nitric oxide synthase (iNOS) induction and activity on delayed protection conferred by ischaemic preconditioning 48 h later in an anaesthetized rabbit model of myocardial infarction. Rabbits underwent a myocardial preconditioning protocol (four 5 min coronary artery occlusions) or were sham-operated. Forty-eight hours later they were subjected to a sustained 30 min coronary occlusion and 120 min reperfusion. Infarct size was determined with triphenyltetrazolium staining. In rabbits receiving no pharmacological intervention, the percentage of myocardium infarcted within the risk zone was 43.9+5.0% in sham-operated animals and this was significantly reduced 48 h after ischaemic preconditioning with four 5 min coronary occlusions to 18.5+5.6% (P<0.01). Administration of the iNOS expression inhibitor dexamethasone (4 mg kg(-1) i.v) 60 min before ischaemic preconditioning completely blocked the infarct-limiting effect of ischaemic preconditioning (infarct size 48.6+/-6.1%). Furthermore, administration of aminoguanidine (300 mg kg(-1), s.c.), a relatively selective inhibitor of iNOS activity, 60 min before sustained ischaemia also abolished the delayed protection afforded by ischaemic preconditioning (infarct size 40.0+/-6.0%). Neither aminoguanidine nor dexamethasone per se had significant effect on myocardial infarct size. Myocardial risk zone volume during coronary ligation, a primary determinant of infarct size in this non-collateralized species, was not significantly different between intervention groups. There were no differences in systolic blood pressure, heart rate, arterial blood pH or rectal temperature between groups throughout the experimental period. These data provide pharmacological evidence that the induction of iNOS, following brief periods of coronary occlusion, is associated with increased myocardial tolerance to infarction 48 h later.

Caspase inhibition and limitation of myocardial infarct size: protection against lethal reperfusion injury.

Ischaemia-reperfusion injury causes cell death by both necrosis and apoptosis. Caspase activation is a major event in apoptosis. We therefore examined the effect of caspase inhibitors during reperfusion upon myocardial infarction. Rat isolated hearts were subjected to 35 min coronary occlusion and 120 min reperfusion. Treatment groups were perfused with caspase inhibitors during early reperfusion. We assessed a non-selective caspase inhibitor (Z-VAD. fmk, 0.1 microM), a caspase-8 inhibitor (Z-IETD.fmk, 0.07 microM), a caspase-9 inhibitor (Z-LEHD.fmk, 0.07 microM) and a caspase-3 inhibitor (Ac-DEVD.cmk, 0.07 microM). All caspase inhibitors limited infarct size (infarct-risk ratio per cent: control 38.5+/-2.6; Z-VAD. fmk 24.6+/-3.4; Z-LEHD.fmk 19.3+/-2.4; Z-IETD.fmk 23.0+/-5.4; Ac-DEVD.cmk 27.8+/-3.3; P<0.05 when compared with control value, 1-way ANOVA). We conclude that caspase inhibition during early reperfusion protects myocardium against lethal reperfusion injury.

Involvement of protein kinase C in the delayed cytoprotection following sublethal ischaemia in rabbit myocardium.

Rabbit hearts were preconditioned with four 5 min coronary artery occlusions 24 h before 30 min coronary occlusion with 120 min reperfusion. Preconditioning significantly reduced the percentage of myocardium infarcting within the risk zone from 49.1 +/- 4.3% to 31.8 +/- 3.5% (P < 0.05). When the protein kinase C (PKC) inhibitor, chelerythrine, was administered just before preconditioning, the delayed protection against infarction 24 h later was abolished. We conclude that the delayed cytoprotective response associated with ischaemic preconditioning of myocardium is likely to involve the early activation of one or more PKC subtypes.

Myocardial protection after monophosphoryl lipid A: studies of delayed anti-ischaemic properties in rabbit heart.

1. Monophosphoryl lipid A (MLA) is a non-pyrogenic derivative of Salmonella lipopolysaccharide. Administration of this agent at high doses to rats and at low doses to dogs was previously shown to confer marked protection against ischaemia-reperfusion 24 h later, although the cellular mechanisms of this delayed protection are obscure. We hypothesized that MLA pretreatment causes the induction of the 70 kDa cytoprotective stress protein HSP70i in the myocardium. If this were the case, protection against ischaemia-reperfusion injury would be observed both in vitro and in vivo. 2. Rabbits were pretreated with MLA 0.035 mg kg-1, i.v. or vehicle solution. For the in vitro study, hearts were isolated 24 h later and Langendorff-perfused with Krebs-Henseleit buffer at 37 degrees C. Global ischaemia was induced for 20 min followed by 120 min reperfusion. Recovery of post-ischaemic left ventricular function and lactate dehydrogenase efflux was similar in MLA and vehicle pretreated hearts and there was no significant difference in the percentage of infarction of the left ventricle determined by triphenyltetrazolium staining (MLA 22.4 +/- 5.2%, vehicle 24.8 +/- 5.1%). 3. When 30 min regional ischaemia and 120 min reperfusion was instituted in pentobarbitone-anaesthetized rabbits 24 h after pretreatment with MLA or vehicle, the percentage infarction within the risk zone was reduced from 42.6 +/- 5.7% in vehicle pretreated animals to 19.6 +/- 4.4% in MLA pretreated animals (P < 0.01). 4. Determination of myocardial HSP70i content by Western blot analysis showed that MLA treatment did not increase HSP70i immunoreactivity. 5. We conclude that MLA at this dose confers protection only against ischaemia-reperfusion injury in vivo and that this protection is not related to induction of HSP70i. Because protection was observed only in vivo it seems possible that the delayed protection conferred by MLA is mediated by effects on humoral or blood-borne factors.

Inhibition by diltiazem of left ventricle collagen proliferation during renovascular hypertension development in rats.

Diltiazem administered in drinking water (0.7 mg mL-1) to Goldblatt two kidney-one clip rats over 16 weeks did not prevent the development of hypertension and left ventricular hypertrophy (LVH). When the collagen content of the left ventricles was assayed (as hydroxyproline), it was found that the fibrosis, characteristic of LVH, was inhibited by diltiazem treatment, despite the fact that hypertension and LVH had developed. This study provides some indirect evidence for the notion that the collagen and myocyte compartments of the myocardium are under separate influences during LVH development in renovascular hypertension.

C-type natriuretic peptide regulation of guanosine-3',5'-cyclic monophosphate production in human endothelial cells.

In vascular smooth muscle cells, relaxant actions of guanosine--3',5'-cyclic monophosphate (cGMP) are well recognized, but there is increasing evidence that cGMP also plays regulatory roles in vascular endothelium. However, the autacoid and endocrine mechanisms controlling cGMP production in endothelium are not well understood. The objective of these studies was to examine the mechanisms of cGMP accumulation in human umbilical vein endothelial cells (HUVEC) in response to natriuretic peptides. Expression in HUVEC of natriuretic peptide receptors, particulate guanylyl cyclases (GC)-A and GC-B, was confirmed by RT-PCR and Western blot analysis. In the presence of the phosphodiesterase inhibitor IBMX 500 microM, 3 h incubation of HUVEC with B-type natriuretic peptide (BNP) (preferential GC-A agonist) or C-type natriuretic peptide (CNP) (preferential GC-B agonist) stimulated concentration-dependent increases in cGMP production. At 10 and 100 nM, we observed two to three-fold greater potency of CNP compared to BNP. In the absence of IBMX, CNP-stimulated cGMP accumulation was significantly less than cGMP accumulation in response to sodium nitroprusside 1 mM. This greater sensitivity of GC-B-derived cGMP to phosphodiesterases suggests compartmentalization of two pools of cGMP from particulate and soluble guanylyl cyclases. Although CNP 100 nM and 1 microM was observed to increase nitrite + nitrate (stable metabolites of NO) production in HUVEC two-fold above basal level, the soluble guanylyl cyclase inhibitor ODQ 10 microM did not significantly modify CNP-stimulated cGMP accumulation suggesting that endothelial actions of CNP may be NO-independent. In conclusion, these studies indicate functional signaling by natriuretic peptides in endothelial cells, supporting possible roles of these mediators in regulating endothelial cell function.