Serum-free culture of rabbit meniscal fibrochondrocytes: proliferative response.

We have formulated a serum-free medium capable of supporting DNA synthesis in rabbit meniscal fibrochondrocytes at a level equivalent to 10% fetal bovine serum (FBS). The medium consists of a 1:1 mixture of Dulbecco's Modified Eagle's Medium and Ham's F-12 medium supplemented with transferrin (1 microgram/ml), selenium (1 pg/ml), trace metal mix (1:100), dexamethasone (100 ng/ml), insulin-like growth factors I and II (50 ng/ml each), pituitary fibroblast growth factor (100 ng/ml), and lactalbumin hydrolysate (2 micrograms/ml). Endothelial cell growth supplement could be substituted for lactalbumin hydrolysate to obtain similar results. Ventrex PC-1, a commercially available, low-protein, serum-free medium, was found to support proliferation of fibrochondrocytes but not as well as 10% FBS or our medium formulation. Lipid supplements, which are known to support the serum-free growth of hyaline chondrocytes, were found to be either of no value or antagonistic for the culture of fibrochondrocytes. Likewise, vitamin E alone, progesterone, putrescine, and hydrocortisone were also without benefit in our culture system. The cells had a more chondrocytic morphology when grown in defined medium as opposed to 10% FBS. The results of this study should now make it possible to identify and quantitate those factors necessary to affect meniscal repair by utilizing further techniques in vitro.

In vitro cell proliferation and proteoglycan synthesis of rabbit meniscal fibrochondrocytes as a function of age and sex.

Meniscal fibrochondrocytes from male and female New Zealand white rabbits, ages 6, 12, and 24 months, were grown in primary and secondary monolayer cell culture. Neither age nor sex affected the majority of their cell culture characteristics. Cells from young males (6 months old) synthesized greater amounts of sulfated proteoglycans than did those from young females, but by 2 years of age, this result was reversed. All age groups synthesized 2 classes of proteoglycans, based on hydrodynamic size, but the ratio of the 2 classes changed as a function of age. Overall, the meniscal fibrochondrocytes from both skeletally immature and skeletally mature rabbits of both sexes were capable of proliferation and matrix synthesis in vitro.

The effects of triethyltin and trimethyltin in rats responding under a DRL schedule of reinforcement.

Rats were trained to respond under a schedule of reinforcement in which only those responses separated by a 10-to 14-sec period of no responding produced a feed pellet (DRL 10 to 14 sec). Each rat received a single dose of trimethyltin (TMT) (5.6, 7.5, or 10 mg/kg) or triethyltin (TET) 1, 3, 4.25, or 5.6 mg/kg). The lowest dose of TMT (5.6 mg/kg) and the lowest dose of TET (1 mg/kg) were without significant effect. At 7.5 mg/kg and 10 mg/kg TMT, the percentage of the total responses spaced 10 to 14 sec apart decreased over the first 8 to 12 days after TMT. Those rats receiving 7.5 mg/kg TMT gradually returned to control values over the next 2 to 3 weeks while those rats receiving 10 mg/kg never recovered. Rats receiving 3, 4.25, and 5.6 mg/kg TET showed a decrease in the percentage of reinforced responses immediately after receiving TET. The behavior of those rats receiving 3 mg/kg returned to control values in 24 hr. Following 4.25 mg/kg TET, the health of the rats deteriorated rapidly. They were kept alive through heroic measures, but then were killed after testing on the 12th day following TET due to their failing health. At 5.6 mg/kg, the rats were killed on the 4th day due to failing health. These results indicate that TMT and TET differ with respect to potency and time course. The behavioral deficits produced by TET parallel the time course of general toxicity while the behavioral effects of acute TMT administration can persist in time long after the general appearance of the rats has returned to normal.

Blockade of ganglionic afterdischarges by tyramine may be mediated by endogenous catecholamines.

Tyramine reduced the compound postganglionic action potential from preganglionic stimulation of the golden hamster isolated stellate ganglion at 0.2 Hz. This action of tyramine was only slightly reduced by phentolamine (10(-5)M), an alpha adrenoceptor antagonist. Tyramine also reduced the postganglionic discharges after preganglionic stimulation at 30 Hz for 2 sec in the presence of hexamethonium. The blockade of afterdischarges by tyramine was markedly reduced by phentolamine. Furthermore, tyramine was much less effective in blocking ganglia from reserpine-pretreated hamsters (6 mg/kg, 4 hr before isolation). Blockade of afterdischarges by norepinephrine was reduced by phentolamine, but not by reserpine. These results indicate that the blockade of afterdischarges with tyramine is mediated by endogenous catecholamines. The blocking action of tyramine was not reduced by cocaine (10(-5)M). Cocaine did reduce the afterdischarges, and this action of cocaine was partially antagonized by phentolamine and reserpine. These results suggest that the blockade of afterdischarges by tyramine and cocaine is due to inhibition of catecholamine uptake which potentiates the actions of endogenous catecholamines.

Interactions of catecholamine uptake inhibitors and norepinephrine on autonomic ganglia.

The effect of catecholamine uptake inhibitors on blockade by norepinephrine was observed in the isolated stellate ganglion of the hamster. The preganglionic nerve was stimulated, supramaximally, at 0.2 Hz and compound action potentials were recorded from the postganglionic nerve. Norepinephrine blocked ganglionic transmission. The sensitivity of the ganglion to norepinephrine was increased by the catecholamine uptake inhibitors, desipramine (3 X 10(-7) M), d-amphetamine (10(-6) and 10(-5) M) and tyramine (10(-4) M). Ouabain (10(-5) and 3 X 10(-5) M) did not change the sensitivity of the ganglion to norepinephrine. All the drugs reduced the uptake by the [3H]norepinephrine into the ganglion. The inhibition of [3H]norepinephrine uptake by the drugs could be correlated with the increase in sensitivity of the ganglion to norepinephrine. These results support the hypothesis that catecholamine uptake is important in terminating the actions of exogenously applied norepinephrine, and that inhibition of the catecholamine uptake increases the sensitivity of the ganglion to norepinephrine.

Potentiation of the ganglionic blocking action of norepinephrine by cocaine.

Concentration-response curves for the blocking action of norepinephrine were determined in the isolated stellate ganglion of the hamster. The blocking effect of norepinephrine on the postganglionic compound action potential from preganglionic nerve stimulation at 0.2 Hz was reduced by phentolamine (10(-5) M), but was not altered by propranolol (10(-6) M). Cocaine (10(-6) and 10(-5) M) increased the sensitivity of the ganglion to norepinephrine. These concentrations of cocaine produced less than 25% blockade when applied alone. The local anesthetic, procaine, did not change the sensitivity of the ganglion to norepinephrine. The blockade by norepinephrine in the presence of cocaine was antagonized by phentolamine and unaffected by propranolol. The time course of the blockade by norepinephrine was more rapid in onset and slower in recovery in the presence of cocaine. If it is assumed that cocaine is blocking norepinephrine uptake into cells within the ganglion, these results indicate that the catecholamine uptake mechanism is involved in terminating the blocking action of exogenously applied norepinephrine.