The in vitro effects of bupivacaine on articular chondrocytes.

We have studied the effects of bupivacaine on human and bovine articular chondrocytes in vitro. Time-lapse confocal microscopy of human articular chondrocytes showed > 95% cellular death after exposure to 0.5% bupivacaine for 30 minutes. Human and bovine chondrocytes exposed to 0.25% bupivacaine had a time-dependent reduction in viability, with longer exposure times resulting in higher cytotoxicity. Cellular death continued even after removal of 0.25% bupivacaine. After exposure to 0.25% bupivacaine for 15 minutes, flow cytometry showed bovine chondrocyte viability to be 41% of saline control after seven days. After exposure to 0.125% bupivacaine for up to 60 minutes, the viability of both bovine and human chondrocytes was similar to that of control groups. These data show that prolonged exposure 0.5% and 0.25% bupivacaine solutions are potentially chondrotoxic.

Interaction of azimilide with neurohumoral and channel receptors.

Binding of the class III antiarrhythmic agent azimilide to brain, heart, and other organ receptors was assessed by standard radioligand binding techniques. In a survey of 60 receptors, azimilide at 10 microM inhibited binding by more than 50% at serotonin uptake (K(i): 0.6 microM), muscarinic (K(i): 0.9 to -3.0 microM), Na(+) channel site 2 (K(i): 4.3 microM), and central sigma (K(i): 6.2 microM) sites. Lesser (20-40%) inhibition was seen at adrenergic, histamine, serotonin, purinergic, angiotensin II, dopamine uptake, and norepinephrine sites and at a voltage-sensitive K(+) channel. In rat ventricle, azimilide inhibited binding to alpha(1)- and beta-adrenergic and muscarinic receptors (K(i): < 5 microM) and to the L-type Ca(2+) channel (K(i): 37.3 microM). In rat brain, azimilide blocked ligand binding to these same receptors and to a serotonin receptor, and the breadth and potency of its interaction pattern differentiated it from ten other class III antiarrhythmics. Azimilide displayed agonist and antagonist action at five muscarinic receptor subtypes in transfected NIH 3T3 cells producing receptor-sensitive mitogenesis and beta-galactosidase activity. Agonist action predominated at M(2) and M(4) subtypes, and antagonist action predominated at M(1), M(3), and M(5) subtypes. The azimilide concentration for 50% maximum stimulation (EC(50)) in M(2)-expressing cells was 1.97 microM (vs 0.14 microM for carbachol). Azimilide's receptor interactions occur at concentrations from one to forty times those required to block cardiac delayed-rectifier channels but could contribute to the efficacy and safety of the drug.

HL-1 cells: a cardiac muscle cell line that contracts and retains phenotypic characteristics of the adult cardiomyocyte.

We have derived a cardiac muscle cell line, designated HL-1, from the AT-1 mouse atrial cardiomyocyte tumor lineage. HL-1 cells can be serially passaged, yet they maintain the ability to contract and retain differentiated cardiac morphological, biochemical, and electrophysiological properties. Ultrastructural characteristics typical of embryonic atrial cardiac muscle cells were found consistently in the cultured HL-1 cells. Reverse transcriptase-PCR-based analyses confirmed a pattern of gene expression similar to that of adult atrial myocytes, including expression of alpha-cardiac myosin heavy chain, alpha-cardiac actin, and connexin43. They also express the gene for atrial natriuretic factor. Immunohistochemical staining of the HL-1 cells indicated that the distribution of the cardiac-specific markers desmin, sarcomeric myosin, and atrial natriuretic factor was similar to that of cultured atrial cardiomyocytes. A delayed rectifier potassium current (IKr) was the most prominent outward current in HL-1 cells. The activating currents displayed inward rectification and deactivating current tails were voltage-dependent, saturated at >>+20 mV, and were highly sensitive to dofetilide (IC50 of 46.9 nM). Specific binding of [3H]dofetilide was saturable and fit a one-site binding isotherm with a Kd of 140 +/- 60 nM and a Bmax of 118 fmol per 10(5) cells. HL-1 cells represent a cardiac myocyte cell line that can be repeatedly passaged and yet maintain a cardiac-specific phenotype.

Pressure- and volume-induced left ventricular hypertrophies are associated with distinct myocyte phenotypes and differential induction of peptide growth factor mRNAs.

BACKGROUND: Chronic pressure and volume overload (PO and VO) result in morphologically and functionally distinct forms of myocardial hypertrophy. We tested the hypothesis that PO- and VO-induced left ventricular (LV) hypertrophies are associated with distinct molecular phenotypes and patterns of peptide growth factor induction. METHODS AND RESULTS: mRNA levels were quantified in LV myocardium from rats with LV hypertrophy due to PO or VO caused by suprarenal aortic constriction or an abdominal aortocaval fistula, respectively, for 1 week. Although PO and VO caused comparable increases in LV weight and preproatrial natriuretic factor mRNA, PO but not VO increased mRNA levels for the fetal genes beta-myosin heavy chain and skeletal alpha-actin and reduced the mRNA level of sarcoplasmic reticulum Ca2+ATPase. In a myocyte-enriched myocardial fraction, transforming growth factor-beta 3 and insulin-like growth factor-1 mRNA levels were increased with PO but not VO; acidic fibroblast growth factor mRNA was unchanged with PO but decreased with VO. In a nonmyocyte-enriched myocardial fraction, transforming growth factor-beta 3 and insulin-like growth factor-1 mRNA levels were decreased with VO but unchanged with PO. CONCLUSIONS: PO- and VO-induced LV hypertrophies are associated with distinct molecular phenotypes and patterns of peptide growth factor induction. Stimulus-specific heterogeneity in the signaling events and peptide growth factors coupled to gene expression could play a role in determining the type of hypertrophy that is caused by various forms of hemodynamic overload.

Hypertrophic stimuli induce transforming growth factor-beta 1 expression in rat ventricular myocytes.

Transforming growth factor-beta 1 (TGF-beta 1) is a peptide growth factor that may play a role in the myocardial response to hypertrophic stimuli. However, the cellular distribution, mechanism of induction, and source of increased TGF-beta 1 in response to hypertrophic stimuli are not known. We tested the hypothesis that the cardiac myocyte responds to hypertrophic stimuli with the increased expression of TGF-beta 1. In adult rat ventricular myocardium freshly dissociated into myocyte and nonmyocyte cellular fractions, the preponderance of TGF-beta 1 mRNA visualized by Northern hybridization was in the nonmyocyte fraction. Abdominal aortic constriction (7 d) and subcutaneous norepinephrine infusion (36 h) each caused ventricular hypertrophy associated with 3.1-fold and 3.8-fold increases, respectively, in TGF-beta 1 mRNA in the myocyte fraction, but had no effect on the level of TGF-beta 1 mRNA in the nonmyocyte fraction. In ventricular myocytes, norepinephrine likewise caused a 4.1-fold increase in TGF-beta 1 mRNA associated with an increase in TGF-beta bioactivity. This induction of TGF-beta 1 mRNA occurred at norepinephrine concentrations as low as 1 nM and was blocked by prazosin, but not propranolol. NE did not increase the TGF-beta 1 mRNA level in nonmyocytes, primarily fibroblasts, cultured from neonatal rat ventricle. Thus, the cardiac myocyte responds to two hypertrophic stimuli, pressure overload and norepinephrine, with the induction of TGF-beta 1. These data support the view that TGF-beta 1, released by myocytes and acting in an autocrine and/or paracrine manner, is involved in myocardial remodeling by hypertrophic stimuli.

Regulation of alpha 1B-adrenergic receptor half-life: protein synthesis dependence and effect of norepinephrine.

Agonist-mediated down-regulation of alpha 1B-adrenergic receptors (AAR) may involve a decrease in synthesis, an increase in degradation, or a combination of the two mechanisms. Norepinephrine (NE) causes downregulation of AAR in rabbit aortic smooth muscle cells (RbSMC). To study the role of receptor degradation in this phenomenon, we studied the half-life (t1/2) of the AAR under basal conditions and during exposure to NE. We first determined the disappearance rate of AAR in the presence of cycloheximide, a commonly used method for measuring receptor t1/2. By this approach, the basal t1/2 was surprisingly long, 172 +/- 20 h. In contrast, the t1/2 in NE-treated cells was 11.8 +/- 0.3 h, suggesting that either NE decreased the t1/2 of AAR or cycloheximide increased the basal t1/2. To distinguish these possibilities, basal receptor t1/2 was determined by a second, independent method based on the repopulation of AAR after irreversible alkylation with chloroethylclonidine. This approach indicated a basal t1/2 (7.4 +/- 0.2 h) that was similar to the t1/2 with NE but more than 20-fold shorter than the t1/2 with cycloheximide. We conclude that in RbSMC NE-induced downregulation of AAR occurs without a decrease in receptor t1/2. The unexpected finding that cycloheximide markedly increases the basal t1/2 of the AAR further indicates that the t1/2 of the AAR is regulated, at least in part, by a short-lived protein that may regulate and/or mediate receptor degradation.

Phorbol esters and norepinephrine destabilize alpha 1B-adrenergic receptor mRNA in vascular smooth muscle cells.

The mechanism by which norepinephrine (NE) down-regulates alpha 1B-adrenergic receptor (alpha-AR) mRNA was studied in rabbit aortic smooth muscle cells. NE, phorbol esters, and bradykinin each decreased alpha-AR mRNA levels by 70-80%. The protein kinase C inhibitor (+)-1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7) abolished the effects of phorbol esters and NE and decreased basal mRNA levels by 52 +/- 3%. Neither ryanodine nor EGTA inhibited down-regulation of alpha-AR mRNA by NE. Actinomycin D caused alpha-AR mRNA level to decrease with a half-life of 3.2 +/- 0.4 h and blocked the effect of H-7 to decrease basal alpha-AR mRNA level. Both NE and phorbol esters increased the rate of alpha-AR mRNA degradation. In NE-desensitized cells, phorbol esters and bradykinin each caused the expected down-regulation of alpha-AR mRNA. The protein phosphatase inhibitor okadaic acid prolonged the normally transient effect of NE for at least 24 h. We conclude that protein kinase C exerts two opposing effects on alpha-AR mRNA levels, 1) a decrease in the stability of the mRNA that requires the sustained phosphorylation of a protein kinase C substrate and 2) a permissive effect on alpha-AR gene transcription.

Alpha 1-adrenergic receptor mRNA level is regulated by norepinephrine in rabbit aortic smooth muscle cells.

Prolonged agonist exposure results in a decrease in the density of alpha 1-adrenergic receptors in rabbit aortic smooth muscle cells. A cDNA for the alpha 1-adrenergic receptor was used to assess the effect of norepinephrine on alpha 1-adrenergic receptor mRNA level in cultured vascular smooth muscle cells from the rabbit aorta. Norepinephrine caused a transient decrease (81% +/- 5%; n = 9) in alpha 1-adrenergic receptor mRNA. The effect was concentration dependent (EC50, approximately 0.3 microM; maximal effect, 10 microM). The maximum decrease occurred after 4 hr of exposure to norepinephrine and was followed by a gradual return to control levels by 24 hr. The decrease in mRNA level was blocked by prazosin, but not propranolol, and was mimicked by phenylephrine. These results indicate that the effect is mediated by stimulation of the alpha 1-adrenergic receptor and suggest that it involves one or more alpha 1-adrenergic-coupled second messenger pathways. The decrease in alpha 1-adrenergic receptor mRNA caused by norepinephrine exceeds that caused by actinomycin D, suggesting that norepinephrine may cause a decrease in the stability of alpha 1-adrenergic receptor mRNA. Actinomycin D also blocked the norepinephrine-induced decrease in mRNA level, further suggesting that the effect of norepinephrine requires induction of transcription, presumably leading to synthesis of a labile factor that is necessary for the effect of norepinephrine on alpha 1-adrenergic receptor mRNA level.

Microcirculatory responses to exogenous endothelial cell-derived relaxing factor.

Endothelium-derived relaxing factor (EDRF) plays an important role in the vasodilatory responses of large blood vessels. However, such a role has yet to be conclusively shown for the microvasculature. In this study we tested the sensitivity of arterioles in the cheek pouch of pentobarbital-anesthetized hamsters to the EDRF-dependent agonists bradykinin and A23187, as well as to exogenous EDRF from cultured bovine aortic endothelial cells. The pouch superfusion fluid was arranged to first pass through a column containing endothelial cells and then on to the tissue. Bradykinin (10-30 nM) or A23187 (0.3 microM) was introduced either upstream or downstream to the endothelial cells, and the resultant responses were measured with video microscopy. Bradykinin and A23187 both caused a dose-dependent release of a microvessel dilator from cultured endothelial cells. We take this dilator to be EDRF based on the characteristics of the responses to the stimuli. Indomethacin (7.7 microM) was present in the superfusate to eliminate the production of cyclooxygenase products from the endothelial cells, and the magnitude of the response was diminished if the superfusate was first passed through a 3-min delay coil before arrival at the pouch. The arterioles dilated to the direct application of bradykinin in a dose-dependent fashion. They did not respond however to the direct application of A23187. These studies demonstrate that arteriolar smooth muscle is able to respond to exogenous EDRF and support the premise that EDRF may play an active role in the regulation of blood flow in the microcirculation.

Endothelin-1 increases intracellular calcium mobilization but not calcium uptake in rabbit vascular smooth muscle cells.

Conflicting evidence has been reported regarding the role of endothelin-1, a potent vasconstrictor peptide, in stimulating extracellular calcium influx in rabbit vascular smooth muscle. The objective of this study was to elucidate the effects of endothelin-1 on transmembrane 45Ca2+ influx and intracellular calcium mobilization in cultured rabbit aortic smooth muscle cells. In calcium containing buffer, endothelin-1 induced a concentration-dependent 45Ca2+ efflux response over the range of 10 pM to 100 nM with an EC50 of approximately 60 pM. Maximum endothelin-stimulated 45Ca2+ efflux was not affected by the absence of extracellular calcium or the presence of 1 microM verapamil. Endothelin-1 did not induce transplasmalemmal 45Ca2+ uptake at times up to 30 min. These findings suggest that an alteration in intracellular calcium handling, rather than extracellular calcium influx, is responsible for the endothelin-stimulated increase in intracellular calcium concentration in rabbit aortic smooth muscle cells.

Endothelium-derived relaxing factor release associated with increased endothelial cell inositol trisphosphate and intracellular calcium.

The release of eicosanoids and endothelium-derived relaxing factor (EDRF) from endothelial cells is thought to involve a calcium-dependent step. Using cultured bovine aortic endothelial cells as a model system, we have examined the relation between agonist-induced changes in inositol polyphosphates and calcium levels within the endothelial cells and extracellular calcium on EDRF release. In a superfusion-cascade system, EDRF was detected by the relaxation of a rabbit aortic ring without endothelium suspended beneath a column of cultured endothelial cells. Endothelial cell stimulation by bradykinin or melittin induced dose-dependent relaxation of the bioassay ring. In addition, bradykinin and melittin stimulated an increase in intracellular calcium concentration in fura-2 loaded endothelial cells and an increase in inositol 1,4,5-trisphosphate (Ins[1,4,5]P3) in cells prelabeled with 3H-myoinositol. Bradykinin stimulation produced transient increases in Ins(1,4,5)P3, fura-2 fluorescence and transient EDRF release. Melittin stimulation induced more prolonged release of EDRF from the endothelial cell column, which was correlated with sustained increases in the fura-2 signal and the level of Ins(1,4,5)P3. Omission of calcium from the cell superfusate attenuated, but did not eliminate, bradykinin-induced EDRF release and the calcium transient, whereas the melittin-induced responses were only slightly attenuated. Endothelial cells clearly demonstrate receptor-activation of phospholipase C and release of sequestered calcium from subcellular sites in response to Ins(1,4,5)P3. These results imply that EDRF release is correlated with increased intracellular calcium levels seen in the absence of extracellular calcium. However, sustained release of EDRF does require influx of extracellular calcium via an undefined mechanism.

Endothelium-dependent relaxation is independent of arachidonic acid release.

Endothelium-dependent relaxation is mediated by the release from vascular endothelium of an endothelium-derived relaxing factor (EDRF). It is not clear what role arachidonic acid has in this process. Inhibition of phospholipase A2, and diacylglycerol lipase in cultured bovine aortic endothelial cells caused a marked reduction in agonist-induced arachidonic acid release from membrane phospholipid pools, and complete inhibition of prostacyclin production. EDRF release, assayed by measuring endothelium-dependent cGMP changes in mixed endothelial-smooth muscle cell cultures, was not inhibited under these conditions. In fact, EDRF release in response to two agonists, melittin and ATP, was actually increased in cells treated with phospholipase A2 inhibitors. In addition, pretreatment of rats with high-dose dexamethasone, an inhibitor of PLA2, did not attenuate endothelium-dependent relaxation in intact aortic rings removed from the animals, or depressor responses in anesthetized animals induced by endothelium-dependent vasodilators. In summary, inhibition of arachidonic acid release from membrane phospholipid pools does not attenuate endothelium-dependent relaxation in rats, or the release and/or response to EDRF in cultured cells.

Use of cultured cells to study the relationship between arachidonic acid and endothelium-derived relaxing factor.

We have used mixed- and co-cultures of endothelial and vascular smooth muscle cells to investigate the role of phospholipase activation and arachidonic acid metabolites in the production of endothelium-derived relaxing factor (EDRF). Inhibition of phospholipase A2 with para-bromophenacyl bromide, dexamethasone or quinacrine, alone or in combination, blocked arachidonate release by 50%-60% but had no effect on EDRF production as assessed by cyclic GMP accumulation in mixed- or co-cultures of endothelial and vascular smooth muscle cells. Inhibition of the phospholipase C-diacylglycerol (DAG) lipase pathway of arachidonate release by the DAG lipase inhibitor RHC-80267 also caused partial inhibition of arachidonate release and had no effect on EDRF. When both phospholipase A2 and phospholipase C pathways for arachidonate mobilization were inhibited (dexamethasone + RHC 80267), arachidonate release was totally inhibited while EDRF release remained intact. We conclude that neither phospholipase activation nor arachidonate mobilization is required for EDRF release from cultured bovine endothelial cells.

Role of calcium in endothelium-dependent relaxation of arterial smooth muscle.

Endothelium-dependent relaxation was studied in rings of rabbit thoracic aorta. Relaxation responses were induced with methacholine, the calcium ionophore A23187 and maitotoxin before and after removal of Ca++ from the external medium; in the presence of calcium-channel entry blockers (verapamil and nifedipine); or with trifluoperazine. Deletion of Ca++ greatly impaired responses to all 3 agonists while trifluoperazine only blocked cholinergic-induced relaxation. The calcium-channel blockers had effects that were concentration- and time-dependent, but their action included blockade of A23187. Cytosolic-free Ca++ concentrations were measured in cultured endothelial cells after incubation of the cells with 10 microM Fura-2/AM or 50 microM Quin 2/AM. Bradykinin (1 X 10(-10) to 1 X 10(-7) M) and melittin (0.5 to 5 micrograms/ml) caused dose-dependent increases in intracellular Ca++ with maximal responses at 3 X 10(-8) M and 3 micrograms/ml, respectively. Both agents were able to induce an increase in cytosolic-free Ca++ in the presence of EGTA (1.5 X 10(-3) M) or verapamil (1 X 10(-5) M). The plateau phase of the Ca++ transient appeared to be modified slightly by verapamil, while the peak responses and plateau were attenuated by '0' Ca++/EGTA. To assess a function of the endothelium, production of endothelium-derived relaxing factor (EDRF) was studied in cells grown on microcarrier beads superfused in a column, and the column effluent was bioassayed on aortic rings.(ABSTRACT TRUNCATED AT 250 WORDS)