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Background We recently demonstrated that donor medial cells are replaced by smooth muscle alpha-actin (SMA)-expressing host cells in murine carotid allografts. In this study, we investigated neointimal cell dynamics including effects of cyclosporine A (CsA) and everolimus (SDZ RAD, Certican). To obtain a functional readout, we measured the effects of these treatments on cardiac allografts. Methods Heterotopic heart allotransplantation was performed between C57BL/6 (B6) and BALB/c (B/c) mice. Orthotopic carotid artery allotransplantation was performed between B6 and B/c mice expressing green fluorescent protein (GFP). Both strains served as donors and recipients. Groups of mice were treated for 4 and 8 weeks (heart and carotid recipients, respectively) with placebo, CsA 20 mg/kg per day, or everolimus 1.0 mg/kg per day using Alzet minipumps. At 2, 4, and 8 weeks, carotid grafts were harvested for histology. Results In the GFP-B/c to B6 strain combination, everolimus but not CsA significantly reduced neointima formation and accumulation of SMA-positive host (non-GFP) cells at doses that did not fully suppress heart rejection. In the B6 to GFP-B/c strain combination, everolimus and CsA strongly prevented heart rejection under treatment, partly suppressed neointima formation, and completely prevented SMA-positive host (GFP) cell accumulation. Conclusions Donor-derived cells expressing SMA never appear in the neointima, and such cells are completely recipient derived. Adequate immunosuppression delays or prevents the disappearance of donor cells and the appearance of host cells in the graft, a phenomenon apparently associated with the neointima formation. Even immunosuppression sufficient to prevent heart allograft rejection under treatment completely, diminishes neointima formation only by approximately 50%.

Background Apolipoprotein E-deficient (apoE-/-) mice spontaneously develop hypercholesterolemia and atherosclerotic lesions. This may be an appropriate background for the development of graft vessel disease. The authors therefore investigated the effects of cyclosporine A (CsA) and SDZ RAD (RAD, everolimus, Certican) on neointima formation of carotid allografts in apoE-/- mice. To ascertain that equipotent immunosuppressive doses were used, the authors also investigated their effects on cardiac allografts using the same wild-type strain combination. Methods Heterotopic heart allotransplantation was performed in the BALB/c to C57BL/6 strain combination. Graft survival was monitored daily. Orthotopic carotid artery allotransplantation was performed from BALB/c to apoE-/- (C57BL/6) mice. Groups of mice were treated for 8 weeks with placebo; CsA 10, 20, and 30 mg/kg/d; or RAD 0.3, 1.0, and 3.0 mg/kg/d using ALZET minipumps. Body weight, CsA blood levels, serum lipids, and histology were examined 8 weeks after transplantation or on the day of rejection. Results Compared with the placebo group, CsA or RAD did not affect body weight. Both CsA and RAD prolonged the survival of cardiac grafts in a dose-dependent manner. CsA blood levels were not different between wild-type and apoE-/- recipients. CsA increased total cholesterol and low-density lipoprotein, but not high-density lipoprotein dose-dependently and significantly, but RAD did not affect the lipids. RAD but not CsA significantly attenuated neointima formation. Conclusions These results suggest that RAD at a dose preventing organ rejection may also prevent transplant vasculopathy even in the presence of hyperlipidemia.

Objective: The macrolide immunosuppressant RAD and the immunomodulator FTY720 have distinct mechanisms of action. We investigated the efficacy of RAD (everolimus, certican®) alone or in combination with FTY720 on graft survival (GS) and histology in comparison with CsA, using mouse strains with strong MHC disparity. Methods: Heterotopic cardiac grafting was performed using the C57Bl/6 to C3H strain combination. Osmotic mini-pumps filled with CsA or RAD were implanted subcutaneously. FTY720 was administered as a single daily dose by gavage. Peripheral lymphocyte count (PLC) was determined at 1, 4 and 8 weeks or on the day of sacrifice. Body weight was recorded on the day of surgery and weekly. Grafts were histologically evaluated. Main findings: In placebo-treated mice the allografts were rejected after 7 days. Monotherapy with 10 and 30 mg/kg/day CsA achieved 10 and 22.5 days median survival time (MST), while 0.1, 0.3, 1 and 3 mg/kg/day RAD resulted in 10.5, 20, >56 and >56 days MST, respectively. FTY720 lowered the PLC significantly, while the lower CsA dose and RAD did not influence the PLC. Adding FTY720 to the 0.6 mg/kg/day dose of RAD extended GS modestly but reduced significantly the perivascular infiltration and endothelialitis in the grafts compared with RAD monotherapy. Conclusions: Under the conditions of the present experiment RAD was more potent than CsA in extending the GS. Combining FTY720 and RAD was well tolerated with respect to weight gain and lack of clinically detectable infections in the mice. The 2-drug regimens suppressed the inflammatory allo-response better than RAD monotherapy.

Background. FTY720 lowers the peripheral lymphocyte count (PLC) by accelerating the migration of circulating lymphocytes to secondary lymphoid organs. We investigated the efficacy of combined FTY720+cyclosporine (CsA) treatment versus monotherapy on prolonging graft survival and on lowering the PLC. Methods. BALB/c hearts were heterotopically grafted in C3H mice. FTY720 was administered alone or in combination with CsA. PLC and body weight were determined on day 7, day 28, or the day of rejection. Results. Combining FTY720 with CsA prolonged, dose-dependently and significantly, the allograft survival. FTY720, but not CsA, lowered the PLC dose-dependently. The granulocyte count was not reduced in any group. FTY720 concentrations were not influenced by the CsA co-administration. Conclusions. Combined FTY720 and CsA treatment was well tolerated, promoted graft survival, and suppressed the inflammatory allo-response. The PLC lowering correlated well with the antirejection effects in the two-drug regimens, suggesting that the PLC might guide FTY720 therapy at low doses.

Background. Graft vessel disease (GVD) is an important problem often responsible for late graft loss. The effects of FTY720, an immunomodulator with a novel mechanism of action were investigated in combination with cyclosporine A (CsA) in a carotid artery allograft model. Methods. A segment of the carotid artery of Lewis rats was replaced by a DA allograft. Seven groups of eight rats were treated for 8 weeks with vehicle (P), CsA 0.3 (C0.3), 1 (C1) or 3 (C3) mgzkg 21 .day 21 or a combination of CsA 1 with FTY 0.01 (C1F0.01), 0.03 (C1F0.03), and 0.1 (C1F0.1) mgzkg 21 .day 21 . Lumen area was estimated by magnetic resonance imaging, peripheral lymphocyte count and drug concentrations were determined at 1 and 8 weeks. Neointima, media, and lumen area were measured morphometrically. Intimal and adventitial infiltration of mononuclear cells, and medial smooth muscle cells number was assessed using a score. Results. FTY720 did not influence CsA blood concentrations. FTY720 but not CsA decreased the PLC dose dependently. Magnetic resonance imaging revealed that treatment groups have larger lumen size than group P. Histological and morphometric evaluation showed that all aspects of GVD were dose dependently suppressed by treatment and lumen narrowing was prevented. Conclusions. CsA, at clinically relevant blood levels, suppressed GVD only partly. The addition of FTY720 was well tolerated and completely suppressed GVD development. In vivo lumen size did not correlate with the histologically estimated neointimal thickness. Graft vessel disease (GVD) remains a major obstacle to long-term graft survival. Perivascular inflammation and concentric neointima formation, combined with medial thinning and necrosis, are observed in 40 ‐ 60% of grafts within the first 5 years of transplantation, frequently resulting in late graft loss (1, 2). Transplanted arteries, like vessels in solid organ grafts, develop concentric and generalized intimal thickening thus differing from classical atherosclerosis (3). Transplantation of blood vessels, especially of the aorta (3‐ 6), can be used to model GVD (7, 8). For mechanistic studies vascular grafts are preferred to solid organ transplants for investigating the specificity of the immunological vessel wall response without the complications of an immune response to parenchymal tissue (8) or parenchymal ischemia and reperfusion damage. We chose carotid artery to represent a small caliber artery reflecting the architecture and fate of smallersized arteries in solid organ transplants that develop intimal thickening, to investigate the suppression of immunologically induced GVD. Our previous investigations have shown that immunosuppression stronger than needed to maintain solid organ grafts, can prevent allograft vasculopathy (7). The main aim of our study was to investigate the contribution of FTY720 added to a low-dose CsA regimen. No results on the effects of FTY720, an agent with a completely new mechanism of action, on GVD have previously been reported. FTY720 [2-amino-2-(2-{4-octylphenyl}ethyl)-1,3-propanediol hydrochloride], is a new compound with a unique mechanism of action characterized by a reduction of the number of circulating lymphocytes. FTY720 accelerates the homing of lymphocytes to peripheral lymph nodes, mesenteric lymph nodes, and Peyer’s patches (9, 10). FTY720 has been shown to extend the survival of rat skin, heart, liver, and small bowel allografts (9, 11‐13), and in combination with CsA also allotransplants in rat, canine, and cynomolgus monkey models (11, 12, 14, 15). The purpose of the study was to analyze the effect of FTY720 in combination with a “reasonable” CsA dose on lesion development in transplanted carotid arteries. We based this approach on our previous experience with studies on lumen size after balloon-catheter injury (16, 17) and after simultaneous allogeneic and autologous transplantation of rat carotid arteries (7). We had observed that histological neointimal thickness did not correlate with in vivo lumen size measured by MRI, which may be functionally more relevant.

Objective: The new immunomodulator 2-amino-2-(2-{4-octylphenyl}ethyl)-1,3-propanediol hydrochloride (FTY720) lowers the peripheral lymphocyte count (PLC) by inducing migration of circulating lymphocytes to secondary lymphoid organs. This effect is dose-dependent at low (up to 0.1 mg/kg per day) doses in rats. We investigated the correlation between PLC and the later rejection, when FTY720 was combined with RAD. Methods: Heterotopic cardiac grafting was performed using the DA–Lewis strain combination. FTY720 and RAD were administered as single daily doses by gavage alone and in combination starting 3 days before to 28 days after transplantation. Graft survival was monitored daily by palpation. PLC was determined at 1 and 4 weeks, body weight (BW) weekly. Histologic evaluation of grafted hearts was performed after rejection. Main findings: FTY720 at doses of 0.03, 0.1 and 0.3 mg/kg per day prolonged graft survival dose-dependently from 6 (placebo) to 7, 9.5 and 15 days median survival time (MST). RAD at doses of 0.3, 1 and 3 mg/kg per day delayed rejection to 8.5, 18 and 37.5 days MST. Very small FTY720 doses added to the lower RAD doses were effective in maintaining grafts throughout the treatment period and with normal weight gain, as opposed to regimens with 1 mg/kg or more per day RAD, which resulted in delayed weight gain. FTY720 lowered the PLC significantly and dose-dependently. The PLC correlated well with graft survival [Spearman rank correlation (n=30, rs=−0.75)]. Conclusions: Fully effective FTY720+RAD combination regimens caused no side effects with respect to the rats’ general well-being or weight gain and were better tolerated than equiactive RAD monotherapy, suggesting a broader therapeutic window for the combinations. Under the experimental conditions, the PLC decrease showed an interesting correlation with the anti-rejection effects in these two-drug regimens. Thus, in rats the PLC is helpful for monitoring the biological activity of FTY720 at low doses (

BACKGROUND: Graft vessel disease is a special form of accelerated arteriosclerosis. Because immunological and nonimmunological factors can contribute to graft vessel disease, we developed a model that enables the study of both factors simultaneously. METHODS: A carotid artery was allografted from DA to Lewis rats, with the excised native artery autografted on the contralateral side. Five groups of six to seven rats were treated for 8 weeks with vehicle (placebo) or cyclosporine (CsA) (0.3, 1, 3, and 10 mg x kg(-1) x day(-1)), which was administered using subcutaneous osmotic minipumps. The carotid lumen area was estimated in vivo at 2, 4, and 8 weeks by magnetic resonance imaging (MRI); CsA blood levels were determined twice. Carotid neointimal thickening and medial and luminal area were measured with histological techniques. RESULTS: MRI showed bulging of the allografts but not autografts. Bulging disappeared over time with narrowing of the allograft lumina estimated by both MRI and histology. Histologically, vehicle-treated animals developed a massive neointima, which was inhibited in a dose-dependent manner by CsA. Autografts remained normal except for minimal subintimal thickening of two of four arteries in the group given the highest dose of CsA. Cellular rejection was detected in the allografts of all but the highest-dose group. The CsA blood levels were similar to those used in man at the two lower doses and about 10-fold higher at the highest dose. CONCLUSIONS: Subintimal thickening did not correlate with in vivo lumen size, a phenomenon that we have previously described for balloon catheter-induced lesions. CsA blood concentrations similar to those used in patients suppressed neointima formation in part, and 10-fold higher concentrations almost completely suppressed neointima formation.

The velocity of blood in the rabbit aorta is very fast, approaching the limits of some pulsed directional Doppler flow meters. Therefore, we thoroughly evaluated a 20- and a 10-MHz device for measuring cardiac output in rabbits. Flow probes were implanted around the ascending aorta and catheters were implanted into the left atrium (for microsphere injection), femoral artery, and vein. About 2 weeks later, cardiac output was determined with the Dopper method and simultaneously with tracer microspheres. Cardiac output was manipulated with isoproterenol, dihydralazine, guanfacine, or alinidine, intravenously. With the 20-MHz device, only normal and decreased cardiac output could be measured accurately, even with a 60° implantation angle of the crystals. The 10MHz device yielded accurate measurements also at very high flow. Surprisingly, at high aortic flow rates, both the 10- and the 20-MHz devices were unable to measure correctly diastolic flow, which is close to zero. It was necessary to adjust the position of the late diastolic Doppler signal manually to the electrical zero line. With this precaution, the 10-MHz device yielded an excellent correlation between mean Doppler signal and cardiac output. Cardiac output can be measured in absolute flow units if the flow-probe can be calibrated in vivo with an independent, accurate method about 2 wk after implantation. The baroreflex was not affected by the implanted flow probes. Within these limits, Doppler flow meters are good tools to assess drug effects on cardiac output in conscious rabbits.

The modification of endothelin effects by the calcium antagonist isradipine was investigated by infusion of 1.0 nM/kg endothelin, a dose known to cause profound vasoconstriction, into eight anesthetized rabbits, followed by an infusion of 10 micrograms/kg isradipine or its vehicle into four rabbits each. In a second series of experiments, only isradipine (same dose) or its vehicle was infused into six rabbits each. Endothelin increased blood pressure and caused systemic vasoconstriction, marked bradycardia, and cardiodepression (measured with a strain guage), yet decreased central venous pressure. The regional vascular effects (measured with microspheres) encompassed widespread vasoconstriction (especially to the kidneys, adrenals, stomach, cecum, and heart), but also a tendency for vasodilatation (hepatic artery). Isradipine decreased blood pressure in endothelin-treated and normal animals. It increased cardiac output more in the endothelin group. The interaction between endothelin and isradipine in the peripheral circulation was generally additive. Isradipine blunts many, but not all, endothelin effects, thus resembling the antivasoconstrictor effects of calcium antagonists against a variety of vasoconstrictor agents such as angiotensin II (Ang II) or vasopressin. Similar to these, endothelin appears to cause vasoconstriction by several mechanisms, one of which apparently involves L-channel activation. A specific interaction of endothelin with the L-channel appears unlikely.

Background. Recent observations have demonstrated the importance of host cells in neointima formation after transplantation. Because little is known regarding the dynamics of host-derived cells in the graft media, we investigated this question in a mouse carotid artery transplantation model. Methods. C57BL/6 carotid arteries were orthotopically transplanted into BALB/c mice ubiquitously expressing enhanced green fluorescent protein. Grafts were harvested at 1, 2, 4, and 8 weeks after transplantation for histologic examination. No immunosuppression was used. Results. Immunostaining and semiquantitative analysis of cross sections showed that donor medial smooth muscle cells decreased over time in the graft media, whereas green fluorescent protein-positive/smooth muscle α-actin-positive cells (i.e., cells of host origin) increased over time. Interestingly, host cells were located only in the inner media and the neointima at 2 weeks and thereafter also in the outer media, indicating that the host-derived cells entered the media from the luminal side rather than from the adventitia. In longitudinal sections, there were no differences in the accumulation of donor- and host-derived cells between the end and middle regions of the graft media at 8 weeks. Conclusions. After transplantation, medial cells were replaced by α-actin–expressing host cells that were probably derived from circulating precursor cells. Our observations differ from the traditional view of a major contribution of donor medial smooth muscle cells to the neointima formation. Thus, circulating progenitor cells may be important for graft vessel disease.

Background. Mycophenolate sodium (MPS), which is in clinical development as an enteric-coated tablet (Myfortic), has not yet been characterized as a combination partner of FTY720. We therefore report here the effects of MPS and FTY720 mono and combination therapy in a rat heart allograft model. Methods. Heterotopic heart allotransplantation was performed in the DA-to-Lewis-strain combinations. Groups of six rats were treated with placebo, MPS monotherapy, FTY720 monotherapy, and their combination. To circumvent pharmacokinetic problems, MPS was administered by Alza minipumps, while FTY720 was administered by gavage, both for 4 weeks. Graft survival was monitored daily, followed up by histology. Body weight and hematological parameters were determined at 1 and 4 weeks. Results. The median survival time (MST) was 6 days for placebo, 6, 14.5, and more than 56 days for doses 3, 10, and 30 mg/kg per day MPS, with severe side effects (diarrhea, weight loss, lymphopenia) at the highest dose, and 7 and 8 days for the 0.03 and 0.1 mg/kg per day FTY720. The combination of 3 mg/kg per day MPS with these two FTY720 doses still resulted in rejection under treatment. However, 10 mg/kg per day MPS in combination with 0.03 or 0.1 mg/kg per day FTY720 yielded MSTs of 41 and 43.5 days, respectively, well beyond the 4-week treatment period. Both combination regimens were well tolerated. Both FTY720 doses combined with the highest MPS dose again caused severe side effects. Conclusions. MPS in monotherapy and combined with FTY720 resulted in steep dose-response curves. However, well tolerated combination regimens could be defined that prevented rejection.

Dimension measurements of the right ventricle are difficult to obtain because of its complex geometry, thus we evaluated a method of right ventricular dimension measurements. Crystals were placed on the ventral and dorsal side of the right ventricular free wall, approximately one-fourth of the right ventricular semicircle away from the septum, in the middle of a cranio-caudal axis of the ventricles. The effects of an increased (infusion of 20 mL/kg of 5% glucose for 3 min into seven rabbits), as well as decreased preload (nitroglycerin, 5 micrograms/kg/min i.v. n = 6) were measured and compared with changes during a placebo infusion (n = 6). The change in shortening of the right ventricle wall segment correlated with changes in both atrial natriuretic factor (ANF) plasma concentration (r = 0.89, p0.05) and central venous pressure (CVP) (r = 0.94, p0.05), respectively. Both these variables are influenced by right ventricular function and dimensions in healthy animals. Dimension measurements obtained across the free wall of the right ventricle appear to reflect right ventricular function well and should be useful in assessing the effects of drugs intended for the treatment of angina pectoris or heart failure.

The severity of myocardial infarction (MI) and its functional consequences are difficult to assess in small animals. We searched for criteria to achieve such an assessment in rabbits 1 week after MI. Thirteen large mongrel rabbits (3-4 kg) were anesthetized with pentobarbitone for ligating a branch of the circumflex coronary artery and 7 rabbits were subject to a sham operation without ligation. All sham-operated rabbits and 12 MI animals survived for 1 week, when blood was obtained for biochemical analyses and the baroreflex was tested. Six animals survived to the third week (survivors) and six died earlier (nonsurvivors). The MI size, measured immediately after death, was 42 +/- 3% of the left ventricular mass in nonsurvivors and 20 +/- 7% in survivors. The plasma atrial natriuretic factor (ANF) concentration was correlated linearly with MI size (r = 0.77) over the whole range of infarct sizes and, like the MI size itself, was associated with the risk of early death (critical limit: 80 pM). Plasma renin activity and catecholamines yielded less prognostic information. The baroreflex control of the heart rate (tested using phenylephrine and nitroprusside) of nonsurvivors was severely impaired and the slopes correlated with MI size (r = 0.90 for phenylephrine and r = 0.67 for nitroprusside). The plasma ANF concentration and the baroreflex both accurately reflected MI size and also correctly classified 11/12 rabbits into survivors and nonsurvivors. An ANF- and baroreflex-based stratification of animals for future studies on therapeutic interventions after MI will reduce the number of animals required by at least 65%, making such studies far more feasible than in the past.

The hemodynamic effects of the calcium antagonist isradipine (code name PN 200-110) and the arteriolar vasodilator dihydralazine were compared in atherosclerotic (cholesterol-fed) and normal conscious rabbits with implanted catheters. Regional blood flows were measured using the microsphere technique. Cardiac output and blood flow to several organs, especially to the gastrointestinal system, but not to the heart and brain, were lower in atherosclerotic rabbits than in normal ones. Intravenous isradipine (10 and 30 micrograms/kg) increased heart rate less in atherosclerotic than in normal rabbits. Isradipine had no effect on the surface electrocardiogram (ECG). In contrast, 0.4 mg/kg dihydralazine caused depression of the ST segment while decreasing blood pressure similarly. This was explained partly by an intramyocardial maldistribution of coronary blood flow (using microspheres). Isradipine increased and dihydralazine decreased flow to the brain. Isradipine redistributed cardiac output in atherosclerotic and normal animals in favor of the heart, brain, and skeletal muscle. However, in atherosclerotic animals, the high dose was less effective than the low dose in some vascular beds. Thus, isradipine is not a general vasodilator in either atherosclerotic or in normal animals, but favors the vital organs. This redistribution of cardiac output contrasts favorably with that induced by dihydralazine.

1. 1. The influence of acute changes in salt balance on the cardiovascular effects of the calcium antagonist isradipine were investigated in two groups of 6 rabbits pretreated 24 hr before an acute experiment, one by substituting 0.45% NaCl for drinking water, the other one with a subcutaneous injection of 20 mg kg−1 furosemide. 2. 2. On the next day the animals were anaesthetized with barbiturates and 10 ml kg−1 0.9% NaCl or 5% glucose were infused into the salt loaded and salt depleted animals respectively. 3. 3. Regional blood flows were then measured with microspheres before and after i.v. administration of 3 and 10 μg kg−1 isradipine (PN200-110). 4. 4. Salt depleted animals had a lower cardiac output and lower flows to several regional vascular beds including the kidneys. 5. 5. The isradipine effects on systemic haemodynamic variables were not different in the two groups. 6. 6. However, the effects on regional blood flows were modified. In salt loaded animals skeletal muscle flow increased more, starting from a higher baseline, yet flow to the kidneys, pancreas and spleen was higher before and decreased significantly more after the calcium antagonist. 7. 7. The results suggest that an alteration in the sodium balance selectively affects the mechanism responsible for vascular tone in a few vascular beds.

Dihydropyridine calcium antagonists such as isradipine (PN200-110) selectively increase blood flow to the heart, brain, and skeletal muscle. The role of the renin-angiotensin system in bringing about this pattern of selectivity was investigated in two groups of eight barbiturate-anesthetized rabbits. One group was and the other one was not pretreated with 1 mg/kg i.v. of the angiotensin converting enzyme inhibitor, spirapril. In the group with the intact renin-angiotensin system, isradipine (3 and 10 micrograms/kg i.v.) elicited the typical systemic and regional (measured with 15 micron tracer microspheres) hemodynamic effects of a noncardiodepressant dihydropyridine derivative. Inhibition of the angiotensin converting enzyme blunted the effects of isradipine on central venous pressure, cardiac output, and flow to the heart, brain, and skeletal muscle. The isradipine-induced decrease of hepatic arterial and pancreatic blood flow was prevented and a similar tendency was observed in the spleen, stomach, small intestine, cecum, and arteriovenous shunt flow, indicating that the renin-angiotensin system was totally (liver, pancreas) or partially responsible for the effects of isradipine on these vessels. The renin-angiotensin system thus counteracts the dilator effect of calcium antagonists in angiotensin II sensitive vascular beds and participates in bringing about the typical pattern of regional vasodilation of these agents.

Calcium entry blockers but not nitroprusside sodium (NPS) or dihydralazine are known to enhance renin release in vitro. Isradipine (PN200-110), NPS or dihydralizine were infused at 15 min invervals into groups of 6 conscious, chronically instrumented rabbits each, at 3 doses carefully matched to elicit comparable falls in blood pressure. Plasma renin activity increased similarly with all treatments. In intact animals, the magnitude of the pressure decrease appears to be more important for renin release than the mechanism of vasodilatation and a typical calcium entry blocker was indistinguishable from other vasodilators.

Tracer microspheres (TM) allow the measurement of regional tissue blood flow. For this purpose the tissue must be cut into the regions selected for measurements. We describe a simple tissue cutter, that greatly facilitates accurate and reproducible cutting of fresh tissue. We have demonstrated its usefulness for obtaining measurements of myocardial blood flow in different layers of the left ventricular free wall of hearts of anesthetized cats and conscious rabbits. Since complete trapping of the TM is essential for blood flow measurements by this method, we investigated the minimal TM size needed by comparing results obtained with different size microspheres. 25 and 15 micrometer TM were trapped equally well by all layers of the left ventricle, but TM smaller than 15 micrometers were not trapped completely. From the size distribution of the 7-10 micrometer TM, we estimated that the functional diameter of the largest capillaries was somewhat smaller (about 7.6 micrometer) in the subepicardial (epi) than in the subendocardial (endo) layer (7.9 micrometer) in both species. When measured with 15 micrometer TM, intramyocardial distribution of blood flow was comparable to that reported for dogs and sheep. In open chest cats, the endo/epi ratio was 1.25 and 1.28, and in conscious rabbits it was 1.35. We conclude that TM with diameters between 10 and 15 micrometers are suitable for measuring intramyocardial blood flow in cats' and rabbits' hearts, and that unlike in dogs and sheep, 7-10 micrometer TM cannot be used.

The stereoisomers of the new dihydropyridine derivative 202-791 [isopropyl 4-(2,1,3-benzoxadiazol-4-yl)-1,4-dihydro-2,6-dimethyl-5-nitro-3 -pyridinecarboxylate] were synthesized separately and tested on isolated rabbit aortic rings for effects on depolarization-induced contraction and depolarization-stimulated uptake of 45Ca2+. The racemic mixture enhanced contraction of rabbit aortic rings at low levels of depolarization but inhibited contraction and 45Ca2+ uptake at high levels of depolarization. The IC50 values were 2.0 X 10(-7) and 1.7 X 10(-7) M, respectively. The R enantiomer inhibited contraction and 45Ca2+ uptake with IC50 values of 3.2 X 10(-8) and 4.3 X 10(-8) M, respectively. This compound showed no stimulant activity. By contrast, the S enantiomer of 202-791 shifted the concentration-response curve for depolarization-induced contraction in an almost parallel fashion to the left, thus enhancing contraction. The EC50 value for this effect at a KCl concentration of 16 mM was 1.8 X 10(-7) M. This compound enhanced 45Ca2+ uptake concentration dependently at all levels of depolarization tested. Thus, the stereoisomers of a dihydropyridine derivative may behave as a calcium entry blocker or a calcium entry enhancer on vascular smooth muscle, depending only on the stereochemistry. If asymmetric compounds elicit effects suggesting a dualistic action at the calcium channel, then the stereoisomers should be prepared.

The effects of a noradrenaline (NA, 1 microgram/kg min) or serotonin (5-HT, 10 micrograms/kg min) infusion on regional blood flow and conductance were assessed with tracer microspheres before and after administration of the calcium-antagonist PY 108-068 (PY; 30 micrograms/kg). Chloralose-urethane-anesthetized cats were pretreated with propranolol 1 mg/kg i.v. or chlorisondamine 1 mg/kg i.v. for the experiments with NA or 5-HT, respectively. PY attenuated the NA-induced increase of blood pressure and the decrease of total peripheral conductance. The NA-induced vasoconstriction, occurring mainly in the kidneys, adrenals, liver, spleen, and arteriovenous shunts, was significantly attenuated by PY. The vessels of heart, stomach, pancreas, and skeletal muscle were not constricted by NA. In contrast, 5-HT dilated or tended to dilate almost all vascular beds examined. Only arteriovenous shunts were constricted. PY did not antagonize this vasoconstrictor effect. Moreover, after but not before PY treatment, a coronary vasoconstrictor effect of 5-HT was seen. Antivasoconstrictor effects of PY were observed both in organs where dihydropyridines normally elicit vasodilatation and in organs where such compounds have no vasodilator effects in the absence of a vasoconstrictor infusion. The regional distribution of the antivasoconstrictor effects was different for each vasoconstrictor tested so far (angiotensin II, 5-HT, and NA). The sites of action of calcium antagonists in peripheral blood vessels appear to depend on the mechanism of activation operating at that site and at the time of the investigation.

DPI 201-106 (DP) is a new cardiotonic agent. In experiment on cats anaesthetized with chloralose/urethane, it lowered blood pressure and caused peripheral vasodilatation at doses between 0.3 and 3 mg/kg, infused i.v. Furthermore, DPI lowered heart rate and increased coronary blood flow (measured with tracer microspheres). The mechanism of this vasodilator action was investigated in experiments in vitro using rabbit aorta. DPI inhibited depolarization-induced contraction. The effect was strongly dependent on the contact time. The pIC50 (-log IC50) values for 15 min, 1, and 2 h pretreatment were 4, 5.2, and 5.7, respectively. 45Ca2+ uptake into rabbit aorta was inhibited by DPI with pIC50 values of 4.3, 5.1, and 5.7, respectively, for the pretreatment periods indicated above. The excellent agreement between effects on tension development and calcium uptake suggests that the mechanism of vasodilatation observed in vivo is related to calcium antagonism.

Peak acceleration of blood in the aorta was measured in chloralose-urethane anesthetized open-chest cats with an electromagnetic flow probe around the aortic root. The effects of substances used in the treatment of chronic congestive heart failure were evaluated in this model. Isoprenaline, noradrenaline, and ouabain increased peak acceleration dose-dependently, while blood pressure, cardiac output, and heart rate were influenced differently by each drug. Nitroglycerin and nitroprusside sodium, afterload reducing agents used for the treatment of congestive heart failure, also increased peak acceleration; an effect that in the case of nitroprusside sodium was shown to be mediated by β-receptors and probably attributable to baroreceptor activation. This simple model was judged to be suitable for evaluating positive inotropic and afterload reducing substances as long as its low sensitivity to heart rate increasing interventions is correctly appreciated.

The enantiomers of PN 200-110 (PN), a highly potent calcium antagonist, were synthesized by a stereoselective synthesis. Cross-contamination was less than 0.2% for the (S)-enantiomer and 0.5% for the (R)-enantiomer. Both isomers inhibited depolarization-induced contraction of rabbit aorta, the (S)-enantiomer being much more active: The pD'2 values were 9.1 and 6.9 for the (S)- and the (R)-enantiomer, respectively. This activity of (R)-PN on potential-operated channels can be attributed to the contamination with 0.5% of (S)-PN. In anesthetized cats, the (S)-enantiomer lowered blood pressure and heart rate dose-dependently (0.3-10 micrograms/kg i.v.). The (R)-enantiomer had almost no effect on heart rate (HR) and blood pressure (BP) at doses up to 300 micrograms/kg. However, both enantiomers increased cardiac output and blood flow to the heart and the brain. The effects on regional blood flow were tissue-dependent, the (R)-enantiomer being surprisingly potent in the subendocardium of the left ventricle. The qualitative and quantitative differences between the activities of the two enantiomers suggest that the results obtained cannot merely be explained by traces of the more active enantiomer contaminating the less active enantiomer. These results, together with those found with the enantiomers of a nitro-substituted dihydropyridine, suggest that calcium channels appear to be able not only to discriminate between enantiomers but also to respond differently to them.

1. 1. The interaction between ergotamine 3 and 30 μg/kg i.v. and darodipine (PY108-068) 30 μg/kg i.v. was studied in anaesthetized cats. 2. 2. Ergotamine decreased heart rate, cardiac output and total peripheral conductance dose-dependently. 3. 3. Ergotamine caused regional vasoconstriction (measured with tracer microspheres) in the heart, kidneys, adrenals, liver, spleen, skin and arterio-venous shunts but dilatation in the pancreas and skeletal muscle, possibly attributable to serotoninergic stimulation. 4. 4. Darodipine reversed most ergotamine effects except those on the spleen, skin and arterio-venous shunts. 5. 5. Since constriction of arterio-venous shunts was not inhibited, ergotamine is unlikely to lose its effectiveness in migraine patients under concomitant dihydropyridine treatment, while unwanted vasoconstrictor effects are likely to be diminished.

The effects of the vasoconstrictive dihydropyridine, CGP 29392, upon tension development and 45Ca2+ uptake by vascular smooth muscle were studied using isolated rabbit aorta. CGP 28392 did not elicit contractile responses when administered alone but lowered the threshold for the contractile response to elevated extracellular K+. CGP 28392 also increased the magnitude of the contractions elicited by submaximal concentrations of K+. This effect was competitively antagonized by PY 108-068, a Ca antagonist of the dihydropyridine class. Similarly, the relaxing effects of PY 108-068 upon KCl-constricted rabbit aorta were competitively antagonized by CGP 28392. The calcium content of unstimulated tissues was only minimally increased by CGP 28392 but the 45Ca2+ uptake stimulated by depolarizing levels of K+ was markedly augmented by this agent. Further analysis of the data indicated that CGP 28392 did not alter the relationship between tension development and 45Ca2+ uptake. In contrast, CGP 28392 was much less effective in augmenting the contractile response and 45Ca2+ uptake elicited by noradrenaline. These results thus support the hypothesis that CGP 28392 predominantly facilitates Ca2+ entry through potential-operated Ca2+ channels.

1. Rabbits were fed a cholesterol-rich diet for 5 two-week intervals. Polyvinyl catheters were then implanted into the femoral artery and vein. Dose-response curves to acetylcholine (ACh), noradrenaline (NA), phenylephrine (Phen) and angiotensin II (AII), were obtained in 6 cholesterol-fed and 6 control rabbits before and after isradipine (code name PN200-110) 100 micrograms kg-1. After these experiments the animals were killed and aortic rings were suspended in an organ bath. ACh but not nitroprusside-induced relaxation was impaired in atherosclerotic but not in control preparations. 2. ACh decreased blood pressure dose-dependently in both groups of rabbits even though ACh did not relax the aortae of the same rabbits in vitro. 3. Blood pressure effects reflect mostly changes in resistance vessels. The pressor effects of NA, Phen and AII were enhanced in atherosclerotic compared with normal rabbits. 4. After a dose of 100 micrograms kg-1 isradipine the dose-response curves of all agents were shifted to the right. The differences between atherosclerotic and control rabbits disappeared, except for the AII-induced pressor response, which remained enhanced in atherosclerotic animals. The calcium antagonist thus only partly corrected the atherosclerosis-associated hyperresponsiveness to vasoconstrictor agents.

Endothelin was infused into seven anaesthetized open-chest rabbits during three 10-min periods, so that cumulative doses of 0.01, 0.1 and 1 nmol/kg were achieved. Endothelin increased blood pressure and caused systemic vasoconstriction only at the highest dose, but decreased the myocardial contractile force (measured with a strain-gauge) at all doses. In contrast to the modest systemic haemodynamic changes, vascular effects (measured with tracer microspheres) were found at each dose level and varied greatly from vasodilator (hepatic artery), through biphasic (spleen) to pure vasoconstrictor (adrenals, pancreas, stomach, colon) effects. Systemic circulatory parameters poorly reflect the regional vascular activity of endothelin.

The new angiotensin converting enzyme inhibitor, spirapril, was found to inhibit the pressor effects of angiotensin I about 10 times more potently than captopril in conscious rabbits. Its effects on systemic hemodynamics and regional blood flows were studied in two groups of 8 normotensive anaesthetized rabbits pretreated 24 h before the acute experiment either by substituting 0.45% NaCl for drinking water or with a subcutaneous injection of 20 mg kg-1 furosemide. Under barbiturate anaesthesia, regional blood flows were then measured with microspheres before and after i.v. administration of two doses of spirapril. With anaesthesia and surgery, plasma renin activity (PRA) was elevated in the salt-replete group and very high in the salt-depleted group (37 and 152 ng ml-1 h-1, respectively). Spirapril lowered blood pressure more in salt-depleted animals and increased only cardiac output and renal blood flow in salt-replete ones. Weak cardiodepression, associated with a small fall of cardiac output, was found in salt-depleted rabbits, indicating that very high angiotensin II levels may contribute to maintain cardiac function in volume-depleted animals with an activated sympathetic system. Coronary and skin blood flow decreased, and pancreatic and hepatic artery flow increased only in this group. A change in the salt balance can thus cause major differences in the responses of the peripheral circulation to angiotensin converting enzyme inhibition.