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Query: UNIPROT:P06889 (Mol)
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Myocytes were isolated by Langendorff perfusion of rat or rabbit hearts with low calcium solution followed by collagenase and hyaluronidase, or by incubation of chunks of rat ventricular tissue in similar media. Cells were then placed in a bath on a microscope stage, superfused and electrically stimulated. Contraction amplitude and rate of change of length during contraction were measured using a video camera and edge detection monitor. Cells were selected for study using a number of criteria developed to identify and define a cell population able to give consistent inotropic responses over a long period. The maximum contraction amplitude with isoproterenol in rabbit cells was 0.244 micron (sarcomere length change) or 13.1% (percentage change in cell length), and the EC50 was 12.8 nM. The maximum contraction amplitude with isoproterenol did not differ significantly between rat and rabbit, between cells prepared by perfusion and those made from chunks, or when determined from non-cumulative rather than cumulative curves. The EC50 for isoproterenol in rat cells made by the perfusion method (cumulative curves) was 3.81 nM, significantly lower than in rabbit. The maximum amplitude obtained with increasing concentrations of calcium was not significantly different from that with isoproterenol under any condition. The EC50 for calcium averaged 2.78 mM in rat cells made by the perfusion method (cumulative curves) and was significantly greater than that in rabbit (1.4 mM). Maximum rates of contraction for rat cells averaged 4.59 micron/s in 8 mM calcium. Rat cells contracted faster than they relaxed, whereas rabbit cells in 8 mM calcium relaxed faster than they contracted. Rat cells, maximally activated by either calcium or isoproterenol, contracted significantly faster than rabbit. There was no difference in rates of contraction (or relaxation) between rat cells prepared by perfusion and those made from chunks of tissue.
J Mol Cell Cardiol 1988 Jul
PMID:Contractile responses of isolated adult rat and rabbit cardiac myocytes to isoproterenol and calcium. 317 50

Using freshly isolated single smooth muscle cells prepared by collagenase treatment, membrane currents were recorded by whole-cell voltage clamp. Intracellular constituents were modified by using an intracellular perfusion technique, i.e., pipette solutions were continuously exchanged from control to test solutions during current recording. In smooth muscle cells, intracellular application of ATP, but not cyclic AMP, enhanced the amplitude of Ca2+ currents and prevented current run-down. In addition, with this stabilization of Ca2+ current recording by ATP, introduction of various chemicals into the cell using the intracellular perfusion technique is useful for investigations of regulation of ion channels in smooth muscle cells.
Mol Cell Biochem
PMID:Whole-cell voltage clamp and intracellular perfusion technique on single smooth muscle cells. 317 40

Regional variations in the size and shape of isolated myocytes were studied using the two-kidney, one clip (2K1C) renal model of hypertension. Weanling male Sprague-Dawley rats (50 to 75 g) were anesthetized by ketamine (100 mg/kg) during renal artery clipping (0.2 mm internal diameter silver clip) and were then allowed to grow for 6 to 8 weeks, when the blood pressure had stabilized at 180 mmHg. Hearts were removed, weighed and then were perfused with a calcium-free Joklik medium containing collagenase. Isolated myocytes were collected from five regions and fixed in isoosmolar glutaraldehyde: right ventricular free wall (RVFW), right and left halves of the interventricular septum (RIVS, LIVS), and epicardial and endocardial halves of the left ventricular free wall (LEPI, LENDO). Myocyte volume was measured by Coulter Counter. Myocyte length was measured by sonic digitizer. Cross-sectional area was calculated from myocyte volume and length. Tailcuff systolic pressure and heart weight were significantly increased in 2K1C rats as compared to control. Body weights were not different. Cell volume was significantly increased in RIVS, LIVS, LEPI, and LENDO, but not in RVFW. Cell length was not significantly increased in any region. Thus, the 2K1C model showed a predominant left ventricular hypertrophy in which the right half of the septum acted in concert with the left ventricle. The shape of the hypertrophied myocytes, having an increase in volume due to an increase in cross-sectional area but not length, was most consistent with a pressure-induced form of cardiac hypertrophy.
J Mol Cell Cardiol 1988 Nov
PMID:Regional myocyte size in two-kidney, one clip renal hypertension. 323 84

A mouse monoclonal antibody (IgG1 isotype) against human C1q (MAb 130) is presented that activates C1 in serum through its antigen-binding sites at an optimal molar ratio of 3 MAbs:1 C1q. The antibody does not inhibit binding of C1q to IgG. Experiments with pepsin- and collagenase-digested C1q showed that MAb 130 binds to the fibril-like strands (arms) of C1q, close to the globular heads. Bivalency of MAb 130 was a requirement for C1-activation, but not for binding to C1q. Increasing the segmental flexibility of the intact antibody by reduction and alkylation destroyed its capacity to activate C1. A MAb against the globular heads of C1q completely inhibited C1-activation by aggregated IgG (AHG), but did not prevent activation by MAb 130. C1, reconstituted by adding C1q-stalks that lack the globular heads to C1q-depleted serum was not activated by AHG, whereas activation by MAb 130 was not affected. Activation of serum-C1 by AHG and MAb 130 was inhibited by addition of excess purified C1-inhibitor in a comparable and dose-dependent manner. Sucrose-gradient analysis indicated a predominance of stable complexes of a single C1q-molecule with three MAbs at the optimal activating ratio. When isolated and added to C1q-depleted serum, these complexes activated C1 efficiently. A mechanism for activation by MAb 130 is proposed that supports the "distortive" model of C1-activation.
Mol Immunol 1988 May
PMID:The distortive mechanism for the activation of complement component C1 supported by studies with a monoclonal antibody against the "arms" of C1q. 326 34

We established culture lines derived from the subendothelial region of the porcine aortic valve. These cells were isolated by extensive collagenase digestion of valvular tissue and were serially propagated with stable morphology. In sparse culture, valve subendothelial cells resembled skin fibroblasts. When confluent, the valve subendothelial cells formed ridges and piles similar to vascular smooth muscle cells. Endogenous in vitro labeling experiments using 35S-methionine showed that valve subendothelial cells synthesized and released several proteins not observed in parallel experiments using porcine skin fibroblasts and smooth muscle cells. Mitogen assays using media conditioned by porcine aortic valvular endothelial cells showed that the valve subendothelial cells, when compared to skin fibroblasts and smooth muscle cells, were particularly avid responders to the growth factors released by valve endothelial cells in vitro. The valve subendothelial cells also released 10-fold more prostacyclin in response to arachidonate than did skin fibroblasts or smooth muscle cells. We conclude that valve subendothelial cells show features that distinguish them from other cultured mesenchymal cells, and that this culture system will be useful for studies of the cellular basis of valvular heart disease.
J Mol Cell Cardiol 1987 Dec
PMID:Porcine cardiac valvular subendothelial cells in culture: cell isolation and growth characteristics. 332 49

A method is described for introducing and expressing cloned genes in isolated hepatocytes. Primary rat hepatocytes isolated by collagenase perfusion were transfected in suspension with plasmid pSV2CAT by electroporation. Forty-eight hours later, soluble extracts from transfected hepatocytes showed chloramphenicol acetyltransferase activity comparable to that obtained in rat hepatoma cell line H4AzC2 by calcium phosphate or DEAE-dextran transfection. The latter two methods could not be used successfully for primary hepatocytes because of cytotoxicity of these reagents. This indicates that electroporation is a useful method to obtain transient expression of foreign genes in primary epithelial cells, such as rat hepatocytes, which are difficult to maintain in cell culture.
Mol Cell Biol 1986 Feb
PMID:Use of electroporation to introduce biologically active foreign genes into primary rat hepatocytes. 346 23

The in vitro effects of ovine PRL (oPRL) on testicular testosterone synthesis were determined using isolated, collagenase-dispersed, adult rat Leydig cells in culture. oPRL (50-1000 ng/ml) had no effect either on basal or on LH (50, 100 or 2000 pg/ml)-stimulated testosterone secretion by Leydig cells in short-term culture (4 h). 125I-oPRL binding studies revealed a single class of high affinity sites (Ka 8.7 nM) with a low capacity (Bmax 6.7 fmol/mg protein identical to approximately 980 sites/Leydig cell). Isolated Leydig cells were further purified on a continuous Percoll gradient and cultured in serum-free medium, at 34 degrees C, in 5% CO2 and 95% air. After 3 days of culture, the media were collected, the cells washed and then stimulated with hCG (3 ng/ml) for 3 h. oPRL (1-1000 ng/ml) added at plating, caused a log dose-dependent inhibition of testosterone accumulation during the 3-day culture period; the highest and most consistent inhibition (31%) was with 500 ng/ml oPRL. hCG increased the sensitivity to the inhibitory effect of PRL, 10 ng/ml oPRL causing 40% inhibition and 100 ng/ml causing a maximal inhibition of 50%. PRL in fact caused a reduction in the maximal effect (efficacy) of hCG on steroidogenesis, without significantly affecting the ED50 (sensitivity). The effects of an antiPRL receptor antibody raised by the antiidiotypic route and previously shown to bind to rat testis PRL receptors were tested. The antiPRL receptor IgG (13 micrograms/ml) mimicked the PRL inhibitory effect and acted synergistically with PRL (100 ng/ml) in inhibiting both testosterone accumulation in 3-day cultured Leydig cells and their subsequent response to hCG. In summary, a clear inhibitory effect of PRL and a synergistic effect of antiPRL receptor antibody were demonstrated on testosterone synthesis by rat Leydig cells in 3-day culture.
Mol Cell Endocrinol 1987 Jul
PMID:Prolactin and antiprolactin receptor antibody inhibit steroidogenesis by purified rat Leydig cells in culture. 362 21

Collagen lysis, which always occurs to some extent in the wound area, is thought to be the underlying cause for breakdown of intestinal anastomoses. Therefore, we have studied the loss of collagen around ileal and colonic anastomoses in New Zealand White rabbits during the first 48 hr after operation. In the ileum, significant lysis of collagen in the anastomotic area, as represented by a decreased level of hydroxyproline, occurs from 12 hr postoperatively onward. Maximal loss of hydroxyproline, as compared to preoperative values, is 27% measured 24 hr after operation. In the colon, significant lysis of collagen occurs after 3 hr. The lowest level of hydroxyproline measured during the experimental period is found 48 hr after operation, where the concentration is decreased by 38%. Changes in ileum are restricted to the anastomotic area, while in the colon the decrease in hydroxyproline extends along the intestinal wall, particularly in a proximal direction. The fact that total protein concentrations do not vary significantly indicates that the lowered hydroxyproline levels are specific. Microscopic examination of the wound area shows that the cellular response during the first 24 hr after wounding is restricted to granulocytes. It is suggested that granulocyte collagenase is mainly responsible for the observed lysis of collagen after intestinal anastomosis.
Exp Mol Pathol 1985 Jun
PMID:Loss of collagen from experimental intestinal anastomoses: early events. 399 58

The role of prolactin on some ovarian functions was studied in collagenase-dispersed luteal cells obtained from PMSG/hCG-primed rats. The in vitro effect of ovine prolactin (oPrl) on luteal cell function was assayed. This hormone produced a dose-dependent increase of progesterone production and an additive effect on hCG stimulation. oPrl had no effect on cAMP production. Chronic effects of prolactin were studied in sulpiride (S), bromocriptine (Br) and oPrl-treated rats. Serum levels of prolactin were significantly higher in S-treated animals whereas Br administration rendered undetectable values. Serum progesterone was reduced in Br-treated animals and LH levels were similar in all groups studied. In vitro studies demonstrated a marked reduction of hCG stimulation of progesterone and cAMP production by luteal cells from hypoprolactinemic animals, while a significant increase was observed in hyperprolactinemic states. oPrl and S treatment significantly increased ovarian LH binding sites while a reduction was observed in Br-treated rats. These data suggest that luteal cell function is regulated by circulating levels of prolactin and that this hormone has some direct effect on the steroidogenic process.
Mol Cell Endocrinol 1984 Jul
PMID:Effect of prolactin on the steroidogenic response of rat luteal cells. 608 23

Mechanical activity in isolated canine atria hinders impalements of single cells with microelectrodes. However, when atrial contractility becomes diminished, a substantial obstacle to impalement remains. To investigate whether the remaining obstacle could be connective tissue, atria were removed from dogs of three age groups (less than 1 year old, 2.8 +/- 0.2 years old and 10.7 +/- 0.5 years old), perfused arterially, and then immersed in elastase (0.01%) or collagenase (0.1%) for 15 min. The following observations were made: (1) In the older atria, satisfactory cell impalements were attained only after they were treated with elastase or collagenase, (2) elastase was effective on the endocardial surfaces only, and (3) collagenase was effective on the epicardial surfaces only. Enzyme effects corresponded to the known anatomic distribution of elastin (endocardial surface) and collagen (epicardial surface). Therefore connective tissue may present a considerable obstacle to cell impalement, especially in hearts from older dogs.
J Mol Cell Cardiol 1984 Sep
PMID:Effects of elastase and collagenase on microelectrode impalements in canine atria at different ages. 609 53


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