Heat acclimation and HSP-72 expression in exercising humans.

The purpose of this research was to determine the effect of a 7-day heat acclimation protocol on HSP-72 expression in human skeletal muscle, and to examine the relationships between molecular and physiological markers of heat acclimation. Ten recreationally active male subjects (age = 23.3 +/- 2.81 yrs, VO(2peak) = 3.85 +/- 0.11 L . min (-1)) completed a 7-day heat acclimation protocol consisting of cycling at 75 % of VO(2peak) in a hot environment (39.5 degrees C, 27 % RH). Muscle biopsies were obtained on days 1 (HTT1) and 7 (HTT2) prior to, 6 h post, and 24 h postexercise to measure HSP-72 protein via SDS-PAGE and silver staining. Core rectal temperatures (T(C)), intramuscular temperatures (T(IM)), skin temperatures (T(SK)), heart rate (HR), oxygen uptake (VO(2)), sweat rate (SR), and plasma cortisol were measured. TC, HR, and plasma cortisol were significantly lower in HTT2 than HTT1 (p < or = 0.05). No significant differences were seen for VO(2), TIM, TSK, or SR when comparing HTT2 with HTT1 (p < or = 0.05). No significant time or day x time interactions were detected for HSP-72 expression (24.48 +/- 2.55 vs. 25.04 +/- 1.43 ng/microg protein for HTT1 and HTT2, respectively, p < or = 0.05). Evidence of heat acclimation was seen at the physiological level; however, no evidence of enhanced thermotolerance at the cellular level was indicated by HSP-72 expression.

Repositioning accuracy of a commercially available double-vacuum whole body immobilization system for stereotactic body radiation therapy.

We evaluated the repositioning accuracy of a commercially available stereotactic whole body immobilization system (BodyFIX, Medical Intelligence, Schwabmuenchen, Germany) in 36 patients treated by hypofractionated stereotactic body radiation therapy. CT data were acquired for positional control of patient and tumor before each fraction of the treatment course. Those control CT datasets were compared with the original treatment planning CT simulation and analyzed with respect to positional misalignment of bony patient anatomy, and the respective position of the treated small lung or liver lesions. We assessed the stereotactic coordinates of distinct bony anatomical landmarks in the original CT and each control dataset. In addition, the target isocenter was recorded in the planning CT simulation dataset. An iterative optimization algorithm was implemented, utilizing a root mean square scoring function to determine the best-fit orientation of subsequent sets of anatomical landmark measurements relative to the original treatment planning CT data set. This allowed for the calculation of the x, y and z-components of translation of the patient's body and the target's center-of-mass for each control CT study, as well as rotation about the principal room axes in the respective CT data sets. In addition to absolute patient/target translation, the total magnitude vector of patient and target misalignment was calculated. A clinical assessment determined whether or not the assigned planning target volume safety margins would have provided the desired target coverage. To this end, each control CT study was co-registered with the original treatment planning study using immobilization system related fiducial markers, and the computed isodose calculation was superimposed. In 109 control setup CT scans available for comparison with their respective treatment planning CT simulation study (2-5 per patient, median 3), anatomical landmark analysis revealed a mean bony landmark translation of -0.4 +/- 3.9 (mean +/- SD), -0.1 +/- 1.6 and 0.3 +/- 3.6 mm in x, y and z-directions, respectively. Bony landmark setup deviations along one or more principal axis larger than 5 mm were observed in 32 control CT studies (29.4%). Body rotations about the x-, y- and z-axis were 0.9 +/- 0.7, 0.8 +/- 0.7 and 1.8 +/- 1.6 degrees, respectively. Assuming a rigid body relationship of target and bony anatomy, the mean computed absolute target translation was 2.9 +/- 3.3, 2.3 +/- 2.5 and 3.2 +/- 2.7 mm in x, y and z-directions, respectively. The median and mean magnitude vector of target isocenter displacement was computed to be 4.9 mm, and 5.7 +/- 3.7 mm. Clinical assessment of PTV/target volume coverage revealed 72 (66.1%), 23 (21.1%), and 14 (12.8%), of excellent (100% isodose coverage), good (>90% isodose coverage), and poor GTV/isodose alignment quality (less than 90% isodose coverage to some aspect of the GTV), respectively. Loss of target volume dose coverage was correlated with translations >5 mm along one or more axes (p<0.0001), rotations >3 degrees about the z-axis (p=0.0007) and body mass index >30 (p<0.0001). The analyzed BodyFIX whole body immobilization system performed favorably compared with other stereotactic body immobilization systems for which peer-reviewed repositioning data exist. While the measured variability in patient and target setup provided clinically acceptable setup accuracy in the vast majority of cases, larger setup deviations were occasional observed. Such deviations constitute a potential for partial target underdosing warranting, in our opinion, a pre-delivery positional assessment procedure (e.g., pre-treatment control CT scan).

The TALON removable head frame system for stereotactic radiosurgery/radiotherapy: measurement of the repositioning accuracy.

PURPOSE: To present the TALON removable head frame system as an immobilization device for single-fraction intensity-modulated stereotactic radiosurgery (IMRS) and fractionated stereotactic intensity-modulated radiotherapy (FS-IMRT); and to evaluate the repositioning accuracy by measurement of anatomic landmark coordinates in repeated computed tomography (CT) examinations. METHODS AND MATERIALS: Nine patients treated by fractionated stereotactic intensity-modulated radiotherapy underwent repeated CTs during their treatment courses. We evaluated anatomic landmark coordinates in a total of 26 repeat CT data sets and respective x, y, and z shifts relative to their positions in the nine treatment-planning reference CTs. An iterative optimization algorithm was employed using a root mean square scoring function to determine the best-fit orientation of subsequent sets of anatomic landmark measurements relative to the original image set. This allowed for the calculation of the x, y, and z components of translation of the target isocenter for each repeat CT. In addition to absolute target isocenter translation, the magnitude (sum vector) of isocenter motion and the patient/target rotation about the three principal axes were calculated. RESULTS: Anatomic landmark analysis over a treatment course of 6 weeks revealed a mean target isocenter translation of 0.95 +/- 0.55, 0.58 +/- 0.46, and 0.51 +/- 0.38 mm in x, y, and z directions, respectively. The mean magnitude of isocenter translation was 1.38 +/- 0.48 mm. The 95% confidence interval ([CI], mean translation plus two standard deviations) for repeated isocenter setup accuracy over the 6-week period was 2.34 mm. Average rotations about the x, y, and z axes were 0.41 +/- 0.36, 0.29 +/- 0.25, and 0.18 +/- 0.15 degrees, respectively. Analysis of the accuracy of the first repeated setup control, representative of single-fraction stereotactic radiosurgery situations, resulted in a mean target isocenter translation in the x, y, and z directions of 0.52 +/- 0.38, 0.56 +/- 0.30, and 0.46 +/- 0.25 mm, respectively. The mean magnitude of isocenter translation was 0.99 +/- 0.28 mm. The 95% confidence interval for these radiosurgery situations was 1.55 mm. Average rotations at first repeated setup control about the x, y, and z axes were 0.24 +/- 0.19, 0.19 +/- 0.17, and 0.19 +/- 0.12 degrees, respectively. CONCLUSION: The TALON relocatable head frame was seen to be well suited for immobilization and repositioning of single-fraction stereotactic radiosurgery treatments. Because of its unique removable design, the system was also seen to provide excellent repeat immobilization and alignment for fractionated stereotactic applications. The exceptional accuracy for the single-fraction stereotactic radiosurgical application of the system was seen to deteriorate only slightly over a 6-week fractionated stereotactic treatment course.

Evidence supporting the Nucleotide Axis Hypothesis: ATP release and metabolism by coronary endothelium.

The Nucleotide Axis Hypothesis, defined and supported herein, proposes that ATP stimulates the release of vasoactive mediators from endothelium, including ATP itself. Here, we show rapid endothelium-dependent, agonist-stimulated ATP elaboration in coronary vessels of guinea pigs. Measurement of extracellular ADP metabolism in intact vessels results in the time- and substrate-dependent formation of ATP in the coronary perfusate in amounts greater than can be accounted for by release from endothelium alone. ATP formation by endothelial cells is saturable (K(M) = 38.5 micromol/l, where K(M) is substrate concentration at which rate is half-maximal.) and trypsin-sensitive, membranes from [gamma-(32)P]ATP-labeled cells support ADP-dependent transphosphorylation by a 20-kDa protein, Western blots reveal the presence of a nucleoside diphosphate kinase (NDPK) of approximately 20 kDa in endothelial membranes, and analysis of NDPK antibody binding by flow cytometry is consistent with the presence of an ecto-NDPK on cardiac endothelial cells. Sequencing of the endothelial cell ecto-NDPK reveals a predicted amino acid sequence with 85% identity to human Nm23-H1 and consistent with a protein whose properties may confer membrane association as well as sites of regulation of activity. Our data underscore the potential importance of a nucleotide axis in cardiac blood vessels.

Comparison of cross-sectional renal function measurements in African Americans with hypertensive nephrosclerosis and of primary formulas to estimate glomerular filtration rate.

Renal function measurements were obtained in 1,703 African Americans with presumed hypertensive nephrosclerosis who were screened for entry into the African-American Study of Hypertension and Kidney Disease (AASK). We examined the effect of race on relationships involving renal variables by comparing African Americans enrolled into the AASK with non-African Americans enrolled into the Modification of Diet in Renal Disease (MDRD) study. We examined the effect of gender on renal variables by comparing African American men and women. We compared various methods for estimating glomerular filtration rate (GFR) with iodine 125-labeled ((125)I)-iothalamate GFR. AASK data were also used to derive a new formula for estimating GFR in African Americans. After adjusting for age, sex, and baseline GFR, African American patients on the AASK study were heavier and had larger body surface areas and body mass indices than either MDRD African Americans or non-African Americans. African Americans had greater serum creatinine levels and urinary creatinine excretions for any given level of GFR. Mean GFR was greater in African American men than African American women (59.7 versus 51.7 mL/min/1.73 m(2)), although serum creatinine levels were also greater in men (1.91 versus 1.73 mg/dL). Seventy-eight percent of women with serum creatinine levels between 1.2 and 1.5 mg/dL had GFRs less than 65 mL/min/1.73 m(2). For African Americans in the AASK, GFR was overestimated by the 24-hour creatinine clearance and underestimated by the Cockcroft-Gault formula. A prediction formula developed in the MDRD study more accurately predicted GFR in AASK patients than these measurements. AASK data were also used to derive a new five-term formula for estimating GFR that was slightly more accurate in the African Americans in the AASK than the MDRD formula (median percentage of error, 12.4% for the MDRD formula versus 12.1% for the AASK formula). Important differences exist in renal variables between African Americans and non-African Americans and between African American men and African American women. Formulas using demographic data and readily measured serum values estimate (125)I-iothalamate GFR.

Effect of ramipril vs amlodipine on renal outcomes in hypertensive nephrosclerosis: a randomized controlled trial.

CONTEXT: Incidence of end-stage renal disease due to hypertension has increased in recent decades, but the optimal strategy for treatment of hypertension to prevent renal failure is unknown, especially among African Americans. OBJECTIVE: To compare the effects of an angiotensin-converting enzyme (ACE) inhibitor (ramipril), a dihydropyridine calcium channel blocker (amlodipine), and a beta-blocker (metoprolol) on hypertensive renal disease progression. DESIGN, SETTING, AND PARTICIPANTS: Interim analysis of a randomized, double-blind, 3 x 2 factorial trial conducted in 1094 African Americans aged 18 to 70 years with hypertensive renal disease (glomerular filtration rate [GFR] of 20-65 mL/min per 1.73 m(2)) enrolled between February 1995 and September 1998. This report compares the ramipril and amlodipine groups following discontinuation of the amlodipine intervention in September 2000. INTERVENTIONS: Participants were randomly assigned to receive amlodipine, 5 to 10 mg/d (n = 217), ramipril, 2.5 to 10 mg/d (n = 436), or metoprolol, 50 to 200 mg/d (n = 441), with other agents added to achieve 1 of 2 blood pressure goals. MAIN OUTCOME MEASURES: The primary outcome measure was the rate of change in GFR; the main secondary outcome was a composite index of the clinical end points of reduction in GFR of more than 50% or 25 mL/min per 1.73 m(2), end-stage renal disease, or death. RESULTS: Among participants with a urinary protein to creatinine ratio of >0.22 (corresponding approximately to proteinuria of more than 300 mg/d), the ramipril group had a 36% (2.02 [SE, 0.74] mL/min per 1.73 m(2)/y) slower mean decline in GFR over 3 years (P =.006) and a 48% reduced risk of the clinical end points vs the amlodipine group (95% confidence interval [CI], 20%-66%). In the entire cohort, there was no significant difference in mean GFR decline from baseline to 3 years between treatment groups (P =.38). However, compared with the amlodipine group, after adjustment for baseline covariates the ramipril group had a 38% reduced risk of clinical end points (95% CI, 13%-56%), a 36% slower mean decline in GFR after 3 months (P =.002), and less proteinuria (P<.001). CONCLUSION: Ramipril, compared with amlodipine, retards renal disease progression in patients with hypertensive renal disease and proteinuria and may offer benefit to patients without proteinuria.

Endothelial cell surface F1-F0 ATP synthase is active in ATP synthesis and is inhibited by angiostatin.

Angiostatin blocks tumor angiogenesis in vivo, almost certainly through its demonstrated ability to block endothelial cell migration and proliferation. Although the mechanism of angiostatin action remains unknown, identification of F(1)-F(O) ATP synthase as the major angiostatin-binding site on the endothelial cell surface suggests that ATP metabolism may play a role in the angiostatin response. Previous studies noting the presence of F(1) ATP synthase subunits on endothelial cells and certain cancer cells did not determine whether this enzyme was functional in ATP synthesis. We now demonstrate that all components of the F(1) ATP synthase catalytic core are present on the endothelial cell surface, where they colocalize into discrete punctate structures. The surface-associated enzyme is active in ATP synthesis as shown by dual-label TLC and bioluminescence assays. Both ATP synthase and ATPase activities of the enzyme are inhibited by angiostatin as well as by antibodies directed against the alpha- and beta-subunits of ATP synthase in cell-based and biochemical assays. Our data suggest that angiostatin inhibits vascularization by suppression of endothelial-surface ATP metabolism, which, in turn, may regulate vascular physiology by established mechanisms. We now have shown that antibodies directed against subunits of ATP synthase exhibit endothelial cell-inhibitory activities comparable to that of angiostatin, indicating that these antibodies function as angiostatin mimetics.

Women and cardiovascular disease.

Cardiovascular disease remains the number one killer in the United States. The face of the person with cardiovascular disease is changing. No longer is the middle-aged man at highest risk. Current data reveal that women, especially postmenopausal women, are at highest risk for cardiovascular disease. This article identifies the differences noted between men and women with cardiovascular disease, including effects in presentation, identification, and treatment.

Pharmacogenomics.

The completion of the Human Genome Project is expected by the year 2003. This mapping of standard human gene sequences and common variants will provide a reference database. This information will also provide new areas of research and study. An example of this is pharmacogenomics, an area of research and development that studies the genetically influenced response to pharmacologic agents.

A historical perspective on the discovery of adenyl purines.

In 1929, Drury and Szent-Gyorgyi described the effects of a simple extract of heart muscle and other tissues on the mammalian heart. This extract was identified as adenylic acid and found to have profound effects on the cardiovascular system. The discovery and identification of adenyl purines and their effects on the cardiovascular system has now extended to other biological functions such as neurotransmission, neuromodulation, and endocrine/exocrine secretory functions and beyond. This review examines the history of the discovery and identification of the many roles played by adenyl purines in regulation of physiological homeostasis.

Role of nucleotides and nucleosides in the regulation of cardiac blood flow.

The regulation of blood flow in the heart on a moment-to-moment basis is essential to meet changes in the oxygen demands of cardiac muscle. The signals that subserve this regulation are not all firmly established. Although the formation and release of adenosine by cardiac muscle during periods of hypoxia or regional ischemia in the heart are well known to produce regional vasodilation and salvage of at-risk myocardium, these extracellular actions of adenosine are believed to occur abluminally and thus do not explain the origin or predict the potent actions of intravascular adenosine. The notion that purines such as adenosine and adenosine 5'-triphosphate (ATP) might be available to act in the lumen of the blood vessel has been proposed by the authors and others to help explain the regulation of blood flow in the heart in nonpathologic states. This article details the background and current understanding of the vascular actions of adenosine and ATP, defines the Nucleotide Axis Hypothesis, and reviews clinical studies in which its likely importance in the maintenance of blood flow in the heart has been investigated.

Nitric oxide and regulation of vascular tone: pharmacological and physiological considerations.

The endothelial cells of the vascular system are responsible for many biological activities that maintain vascular homeostasis. Responding to a variety of chemical and physical stimuli, the endothelium elaborates a host of vasoactive agents. One of these agents, endothelium-derived relaxing factor, now accepted as nitric oxide, influences both cellular constituents of the blood and vascular smooth muscle. A principal intracellular target for nitric oxide is guanylate cyclase, which, when activated, increases the intracellular concentration of cyclic guanosine monophosphate, which in turn activates protein kinase G. Acting by this pathway, nitric oxide induces relaxation of vascular smooth muscle and inhibits platelet activation and aggregation. Derangements in endothelial production of nitric oxide are implicated as both cause and consequence of vascular diseases, including hypertension, atherosclerosis, and coronary artery disease.

What's spirit got to do with it? Interview by Valerie Michele Hoskins.

AIDS: HIV-infected people are turning to strengthening the spirit while fighting the virus. Spiritual leaders participated in a discussion about the impact of spirituality on people living with HIV and responded to such questions as the relationship between the spirituality-seeking patient and the spiritual leader, whether spiritual support has grown or decreased with the advent of protease inhibitors, and whether the alternative therapies used today are considered spiritual tools. Participants also discussed rituals or practices they feel should be used to attain a healthy mind, body, and spirit; other appropriate services for HIV-positive people; and obstacles to more spiritual and religious resources sensitive and responsive to HIV-positive people.^ieng

Competition of adenine nucleotides for a 1,3-[3H]-dipropyl-8-cyclopentylxanthine binding site in rat vas deferens.

1. The binding of 1,3-[3H]-dipropyl-8-cyclopentylxanthine ([3H]-DPCPX), a specific adenosine A1 receptor antagonist, was examined in rat vas deferens membrane preparations using radioligand binding techniques. 2. 1,3-[3H]-Dipropyl-8-cyclopentylxanthine bound to these preparations with a KD of 1.07 +/- 0.14 nmol/L (n = 6). The density of [3H]-DPCPX binding sites was 133.38 +/- 5.57 fmol/mg protein. 3. Computer analysis indicated that nucleosides competed for [3H]-DPCPX binding at two distinct sites. The rank order of potency at the higher affinity site corresponded to R-phenylisopropyladenosine (R-PIA) > or = 2-chloroadenosine (2-CIADO) > or = cyclopentyladenosine (CPA) > or = N-ethylcarboxamidoadenosine (NECA) > s-phenylisopropyladenosine (s-PIA). Ki values were in the low nmol/L range. The rank order of nucleoside potency at the lower affinity site corresponded to R-PIA > or = CPA > or = NECA > or = 2-CIADO > S-PIA. Ki values were in the low mumol/L range. 4. Nucleotides competed for [3H]-DPCPX binding at a single site only. The rank order of potency at this site corresponded to alpha, beta-methylene ATP > or = beta, gamma-methylene ATP > or = ATP. Ki values were in the high mumol/L range. The site seemed to correspond with one of the two binding sites predicted by nucleoside competition binding. 5. The ATP-regenerating compound myokinase did not significantly change the competition curve for ATP, indicating that the competition for [3H]-DPCPX binding observed in the presence of ATP was due to an effect of ATP per se and not to an action of a degradation product. 6. The results demonstrate that in rat vasa deferentia there exist two distinct binding sites for [3H]-DPCPX. One of these sites binds only nucleosides and may represent an adenosine A1 receptor, as usually defined. The other site binds both nucleosides and nucleotides and may represent an atypical adenosine A1 receptor, an atypical P2 or a P3 purinoceptor.

Calciphylaxis in chronic renal failure.

Calciphylaxis is a rare and life-threatening complication that is estimated to occur in 1% of patients with ESRD each year. Typically, extensive microvascular calcification and occlusion/thrombosis leads to violaceous skin lesions, which progress to nonhealing ulcers and sepsis. Secondary infection of skin lesions is common, often leading to sepsis and death. The lower extremities are predominantly involved (roughly 90% of patients). Patients with skin involvement over the trunk or proximal extremities have a poorer prognosis. Although most calciphylaxis patients have abnormalities of the calcium:phosphate axis or elevated levels of parathyroid hormone, these abnormalities do not appear to be fundamental to the pathophysiology of the disorder, and the etiology of calciphylaxis remains unclear. Recently, functional protein C deficiency has been hypothesized to cause a hypercoagulable state that could induce thrombosis in small vessels, with resulting skin ischemia, necrosis, and gangrene. The lack of understanding of the pathophysiology of the disease results in treatments that are equally unsatisfactory. Patients who undergo parathyroidectomy have a tendency to improve, but the prognosis for the disease is poor and mortality remains high.

Molecular analysis of a metalloprotease from Proteus mirabilis.

Proteus mirabilis is known for its ability to differentiate from swimmer to swarmer cells, a process crucial for the pathogenesis of these bacteria during urinary tract infections. Among the many virulence factors produced during swarmer cell differentiation is an extracellular metalloprotease. A cosmid containing a large fragment of P. mirabilis chromosomal DNA was obtained by measuring protease expression in recombinant Escherichia coli. The recombinant and native enzymes were purified to over 95% homogeneity from culture supernatants by use of phenyl-Sepharose affinity chromatography and found to be identical. The activity of the 55-kDa enzyme was stimulated by divalent cations (Ca2+ > Mg2+) and inhibited by a chelator of these cations. The enzyme possesses substrate specificity for both serum and secretory forms of immunoglobulin A1 (IgA1) and IgA2 as well as IgG and, unlike classic IgA proteases, digested to completion both human and mouse IgA. Following subcloning, a 5-kb DNA fragment encoding recombinant protease activity was identified by insertional mutagenesis with Tn5. Four open reading frames were identified within this 5-kb region by limited nucleotide sequence analysis of DNA flanking the transposon. The nucleotide and deduced amino acid sequences of the metalloprotease structural gene (zapA) were obtained. Computerized homology studies revealed that the P. mirabilis metalloprotein is a member of the serralysin family of proteases and may be part of an operon comprising genes encoding an ATP-dependent ABC transporter in addition to the metalloprotease. The relevance of the metalloprotease to swarmer cell differentiation and pathogenicity is discussed.

Purinergic axis in cardiac blood vessels. Agonist-mediated release of ATP from cardiac endothelial cells.

Purified endothelial cells isolated from guinea pig hearts by enzymatic perfusion were grown in monolayer culture and used to test the ability of a variety of vasoactive agents to stimulate ATP release from these cells. Stimulation of endothelial cells with the peptide agonist bradykinin (1 nmol/L), acetylcholine (1 mumol/L), serotonin (1 mumol/L), or adenosine 5'-diphosphate (10 mumol/L) resulted in the rapid appearance of ATP in the incubation medium determined with the firefly luciferase assay for ATP. Addition of antagonists for muscarinic (atropine, 0.1 mumol/L) and purinergic (suramin, 100 mumol/L; reactive blue-2, 100 mumol/L) receptors suggested that ATP release from these cells was receptor-mediated. Bradykinin-induced release of ATP was rapid (peak < 30 seconds at 3 nmol/L bradykinin), dose-dependent (EC50, 0.18 nmol/L), and diminished with repeated administration of agonist. Desensitization to bradykinin also affected the ability of acetylcholine to induce release and was reversible when cells were returned to growth conditions for short periods. Measurement of released adenyl purines as their fluorescent N6-ethenopurine derivatives by high-performance liquid chromatography revealed the origin of the purine released to be ATP and confirmed its rapid dephosphorylation. Addition of the purine nucleotide analogues 2-methylthio-ATP (2-methyl-S-ATP), ADP, and beta gamma-methylene ATP to endothelial cell cultures resulted in a dose-dependent increase in the appearance of ATP measured in the medium bathing the cells at 30 seconds, suggesting the presence of ATP-induced ATP release.(ABSTRACT TRUNCATED AT 250 WORDS)

NG-nitro L-arginine methyl ester and other alkyl esters of arginine are muscarinic receptor antagonists.

Analogues of L-arginine with modifications at the terminal guanidino nitrogen and/or the carboxyl terminus of the molecule have been widely used for their ability to inhibit the production of nitric oxide and are thought to be competitive antagonists of nitric oxide synthase. The present studies were designed to test the possibility that these agents are also muscarinic receptor antagonists. Acetylcholine produced concentration-dependent contraction of endothelium-denuded rabbit coronary artery as well as isolated strips of canine colonic smooth muscle. The arginine analogue NG-nitro L-arginine methyl ester (L-NAME, 100 microM) but not NG-monomethyl L-arginine (L-NMMA, 100 microM) significantly shifted these contractile relations to the right, an effect that was not reversed by addition of 1 mM L-arginine. In radioligand binding studies using the muscarinic radioligand [3H]quinuclidinyl benzilate and tissues known to contain differing contributions of M1, M2, and M3 muscarinic receptors, addition of increasing concentrations of L-NAME resulted in a monophasic competition of binding with affinities (Ki) ranging from 68 microM in endothelium to 317 microM in whole aorta. Addition of the hydrolysis-resistant guanosine 5'-triphosphate analogue GTP gamma S (100 microM) had no effect on L-NAME competition of [3H]quinuclidinyl benzilate binding. Addition of L-NAME in radioligand binding competition studies using the agonist carbachol did not result in an alteration of the receptor's affinity for agonist, confirming the competitive nature of the interaction of L-NAME with the muscarinic receptor.(ABSTRACT TRUNCATED AT 250 WORDS)