Presence of luteinizing hormone-releasing hormone binding sites in cultured porcine lymphocytes.

Membrane-enriched homogenates of fresh and cultured (48 h) porcine lymphocytes were assayed for the presence of specific LHRH receptors by saturation and displacement analysis using [D-Ser-(TBU)6, des-Gly-NH2(10)] LHRH-EA as the labeled and unlabeled ligand. Membrane-enriched homogenates of porcine pituitaries served as positive controls while porcine granulosa cell membranes and crude liver homogenates served as negative controls. Specific high-affinity LHRH receptors were found in porcine pituitaries (Kd = 0.3 nM) and cultured lymphocytes (Kd = 13 nM) but not in fresh lymphocytes. No specific binding was observed in negative control tissues. Porcine lymphocytes have measurable high-affinity LHRH receptors after 48 h of culture.

Kinetic characteristics of beta-carotene uptake after an injection of beta-carotene in pigs.

Two experiments were conducted to study the uptake of beta-carotene in plasma, lipoproteins, and blood cells in pigs (50 to 55 kg; n = 40) after an i.m. injection of 0, 10, 20, or 40 mg of beta-carotene. Blood was sampled at 0, 3, 6, 12, 24, and 48 h postinjection. beta-Carotene was not detectable in plasma, lipoproteins, or blood cells of control pigs. However, concentrations of beta-carotene in plasma and lipoproteins increased in a dose-related manner in injected animals. Distribution of beta-carotene in the lipoproteins changed with time postinjection. The beta-carotene associated with very low density lipoproteins increased and that in low density lipoproteins decreased with time in treated pigs. Concentrations of beta-carotene in lymphocytes of treated pigs also increased within 3 h postinjection. The profile of beta-carotene in lymphocytes was different from that observed in plasma and lipoproteins. Carotene was not detectable in neutrophils and erythrocytes. Treatment did not alter concentrations of retinol or alpha-tocopherol in plasma, lipoproteins, or blood cells. Therefore, lymphocytes specifically take up beta-carotene, thereby suggesting a possible role of beta-carotene in this immune cell.

Subcellular distribution of beta-carotene, retinol, and alpha-tocopherol in porcine lymphocytes after a single injection of beta-carotene.

The subcellular distribution of beta-carotene, retinol, and alpha-tocopherol in lymphocytes was studied in pigs (50 to 55 kg) injected once with 0, 20, or 40 mg of beta-carotene. Blood was sampled at 0, 24, 48, and 72 h postinjection. Plasma beta-carotene in treated pigs peaked at 24 h and decreased rapidly thereafter. Beta-carotene was found in all subcellular fractions of lymphocytes. Concentrations in nuclei mirrored changes in plasma. However, beta-carotene in mitochondria and cytosol peaked at 24 h, whereas that in microsomes peaked at 48 h. Concentrations in the latter three subcellular fractions remained high at 48 and 72 h even though plasma beta-carotene had decreased to very low concentrations. Peak concentrations of beta-carotene were highest in the nuclei, intermediate in the mitochondria and microsomes, and lowest in the cytosol. Treatment did not influence concentrations of retinol or alpha-tocopherol in the various subcellular fractions. These data provide more compelling evidence for the possible role of beta-carotene in lymphocytes.

Quantitative and qualitative changes in blood leukocytes in the porcine ovary.

The distribution of blood leukocytes in the preovulatory follicle (PO) and in the corpus luteum (CL) of the pig was studied by light microscopy. The number of macrophages increased in the freshly luteinized follicle, decreased subsequently in the developing and mature CL, and then increased again in the regressing CL. However, the relative proportion of macrophages as a percent of total blood leukocytes present did not change throughout the cycle. The increase in the number of lymphocytes was the greatest increase after ovulation as compared to all the other blood leukocytes observed. Even though the number of lymphocytes decreased thereafter, their numbers expressed as a percent of total blood leukocyte number remained high in the developing and mature CL. Eosinophils were the most prominent blood cell type present in thecal tissue of PO and in the regressing CL. In other stages of the CL, few eosinophils were observed. Neutrophil numbers remained moderate and unchanged throughout most of the estrous cycle except in the regressing CL, where the number of neutrophils was slightly increased. Low numbers of plasma cells were observed in all structures studied and no significant changes due to stage were apparent. The distribution of lymphocytes in the corpus hemorrhagicum (CH) and eosinophils in the regressing CL was different in different regions of the ovarian structure. In summary, blood leukocytes, most notably macrophages, lymphocytes and eosinophils, differentially migrate into specific structures of the ovary at specific stages of the estrous cycle of the pig. The possible involvement of these blood leukocytes in the modulation of ovarian events is discussed.

Porcine lymphocytes secrete a bioactive and immunoreactive LH-like factor in response to LHRH and concanavalin A.

Lymphocyte conditioned medium (CM) was prepared by incubating 0 (cell-free control) or 4 x 10(6) lymphocytes/ml in serum-supplemented RPMI containing 0, 10(-9), 10(-7), and 10(-5) M luteinizing hormone releasing hormone (LHRH), 10(-5) M LHRH antagonist (LHRHA), or 10(-7) M LHRH + 10(-5) M LHRHA. Treatments were applied with and without 10 micrograms/ml concanavalin A (con A), and media were analyzed for LH. Aliquots of the CM from cultures incubated for 48 h were later applied to porcine granulosa cell cultures (suspended to 1.25 x 10(5) cells in 450 mul). Thereafter, 50 mul of CM were added to granulosa cell cultures. Media were collected after 12, 24, and 48 h and progesterone determined. Immunoreactive LH increased with time of incubation in lymphocyte CM but not cell-free CM. LH content of lymphocyte CM increased as LHRH concentration increased. LHRHA significantly reduced the amount of LH measured. The presence of con A in the medium resulted in maximal concentrations of LH, irrespective of dose of LHRH or LHRHA. Cell-free CM containing LHRH, LHRHA, and/or con A did not affect progesterone production by granulosa cells at any of the time periods. Lymphocyte CM containing LHRH caused a dose-dependent increase in progesterone production at 48 h. This stimulation was blocked by lymphocyte CM containing LHRHA. Lymphocyte CM containing con A also stimulated progesterone production at all of the LHRH concentrations studied. This response was not inhibited by lymphocyte CM containing the LHRHA.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of blood monocytes and lymphocytes on progesterone production by granulosa cells from small and large follicles in the pig.

The objectives of this study were 1) to determine the influence of lymphocytes and monocytes on progesterone secretion by porcine granulosa cells in culture, and 2) to compare the responses of granulosa cells obtained from small versus large follicles. Granulosa cells were cocultured with lymphocytes, monocytes, and unfractionated mononuclear cells in serum-free culture medium. Progesterone content of media and cellular DNA were determined after 6, 12, and 24 hrs of culture. Progesterone production by granulosa cells alone or in coculture with erythrocytes increased (P less than .001) up to 12 hrs and plateaued thereafter. Cells from large follicles produced approximately two-fold (P less than .01) more progesterone as compared to those isolated from small follicles. Cell numbers, as indicated by DNA, did not change with time in any of the cultures. Lymphocytes stimulated (P less than .05) progesterone production by granulosa cells from large follicles but not from small follicles. Similar results were seen when unfractionated mononuclear cells were cocultured with granulosa cells. Monocytes stimulated progesterone production by granulosa cells from both large and small follicles. Lymphocytes and monocytes influence steroid secretion by granulosa cells in the pig in vitro. These effects are not mediated through cell proliferation and are influenced by follicle size.

Absorption and distribution of xenobiotics.

Extrapolation of pharmacokinetic data between species has been simplified by the advent of more sensitive methods of analysis of chemicals in body tissues and by the capability of inexpensive computers to perform complex calculations. These new methods enable investigators to observe the rates at which target tissues reach equilibrium in different species and to develop mathematical models of these processes. The evaluation of physiological pharmacokinetics from classical or compartmental kinetics is improving the ability to project the long-term behavior of chemicals in body fluids and organs based on independently derived physical, chemical, and physiological constants obtained from simple chemical reactions, tissue culture experiments, or short-term animal studies. Accurate prediction of chemical behavior by such models gives support to hypothetical mechanisms of distribution and accumulation, while significant deviations from predicted behavior signal the existence of previously unsuspected pathways. These techniques permit the simulation of the impact of linear, nonlinear, and saturation kinetics on chemical behavior; the prediction of integrated tissue exposure; and the mapping of the sequence of alternate metabolic pathways that lead to toxicity or detoxification. The discussion will identify the research needs for improving extrapolations between species.

Motor nerve ending disorder in myasthenia gravis.

Mild myasthenia gravis patients were compared with normals with respect to the capacity of their motor nerve endings (MNEs) to generate a neostigmine-induced postactivation repetition (PAR). Dose-response analyses of PAR recorded from muscle electrically and by contractile measurement disclose a loss of this pharmacologic responsiveness in myasthenia. Since mild myasthenics transmitted nerve impulse trains of 20 to 200 Hz, as did normals, it was evident that PAR is transmitted insofar as it can be generated by MNEs. The dose-response analyses support this. These data indicate an MNE disorder in the disease.

Physostigmine inhibition of 3',5'-cyclic AMP phosphodiesterase from cat sciatic nerve.

This study was designed to determine whether cholinergic drug interaction with cyclic (c) AMP phosphodiesterase (PDE) might account for part of the effects of this class of drugs at the neuromuscular junction. The activity levels of both high- and low-affinity forms of cAMP PDE from cat sciatic nerve were examined for drug inhibition or activation. Of the cholinergic drugs tested, only physostigmine and Tacrine produced significant cAMP PDE inhibition. Physostigmine was 10 times more potent than theophylline and half as potent as SQ 20,009 (known PDE inhibitors) in inhibiting motor nerve cAMP PDE. Tacrine inhibited this enzyme at concentrations comparable with theophylline. None of the other drugs tested (diisopropylfluorophosphate, edrophonium, neostigmine, ecothiophate, carbachol or d-tubocurarine) produced significant changes in cAMP PDE activity. The inhibitory effects of physostigmine were shown to be pH independent over a range of 7.0 to 8.5. Kinetic studies indicated a mixed form of inhibition for physostigmine and Tacrine comparable with that seen for theophylline. These data indicate that the anticholinesterase activity of physostigmine and Tacrine do not adequately describe the facilitatory actions of these drugs at the motor nerve ending.

Characterization of the train-of-four response in fast and slow muscles: effect of d-tubocurarine, pancuronium, and vecuronium.

The in vivo cat soleus and gastrocnemius muscles were used to compare isometric contraction strength and the train-of-four (T4) response (2 Hz for 2 s) of two muscle types (fast and slow) during onset of competitive neuromuscular blockade in order to determine the extent of the correlation between twitch depression and T4 fade. Prior to drug administration the muscles that were studied differed significantly in that the T4 ratio was 1.0 in the gastrocnemius and only 0.87 in the soleus. Three competitive neuromuscular-blocking agents were compared: d-tubocurarine, pancuronium, and vecuronium. d-Tubocurarine was found to produce a close correlation between the degrees of twitch strength depression and T4 for both muscles. However, these muscles demonstrated significantly different ED50 values (105 micrograms/kg for gastrocnemius, 150 micrograms/kg for soleus). Pancuronium also produced a similar relationship between twitch strength depression and T4 decrement for each muscle. In this case, however, there was little difference in their ED50 values for twitch depression (11.5 micrograms/kg for gastrocnemius, 13 micrograms/kg for soleus). The effects of vecuronium were quite different from the other two muscle relaxants. Although vecuronium produced a comparable correlation between twitch tension and T4 fade in fast muscle, no such relationship was found to exist in slow muscle. Even when the twitch strength was blocked to 18% of control, the soleus T4 response was depressed to only 75% of control. These results highlight major differences among competitive neuromuscular-blocking agents and suggest multiple sites of action.

Cyclic nucleotides and neuromuscular transmission.

A review of the research on cyclic nucleotides and neuromuscular transmission suggests that cAMP is involved in the release of transmitter from motor nerve endings. Lipid-soluble derivations of cAMP cause depolarization of unstimulated nerve endings and prolong the after potentials of stimulated nerve endings. They also increase the frequency of miniature end plate potentials and increase the quantal content of stimulus evoked end plate potentials. Similar effects are produced by compounds that activate adenylate cyclase or inhibit phosphodiesterase. The responses to the derivatives of cAMP and activators of cyclase are enhanced by inhibitors of phosphodiesterase and prevented by compounds that block the flux of calcium into nerve endings. There is no evidence that suggests that cyclic nucleotides are involved in the postjunctional response to transmitter. Thus, it seems likely that cAMP is involved in the regulation of calcium in motor nerve endings and the exocytosis of transmitter. Additional study should expand our knowledge of neuromuscular transmission and contribute to an understanding of the functions of cyclic nucleotides in other synapses.

Effects of calcium on a cyclic nucleotide system in soleus motor nerve terminals.

The effects of increased extracellular calcium levels on the responses of the motor nerve terminal to agents that increased intracellular levels of cyclic AMP was studied on the in vivo cat soleus muscle preparation. The neural and muscle responses to intra-arterially administered NaF, an activator of adenylate cyclase, and dbcAMP progressively increased as blood calcium levels rose. These results provide additional support for the hypothesis of an interaction between calcium and cyclic AMP in motor nerve endings.