Correlation between erythropoietic activity and body growth rate in hypertransfused polycythemic growing rats as the result of an erythropoietin-dependent operating mechanism.

The established relationship between erythropoietic activity and body growth rate in the polycythemic growing rat could be the result of either an erythropoietin (EPO)-dependent or an EPO-independent operating mechanism. The present study was thus undertaken to elucidate the nature of the aforementioned mechanism by assessing the ratio between plasma immunoreactive EPO (iEPO) concentration and erythropoietic activity in young hypertransfused rats for different body growth rates. Red blood cell (RBC)-59Fe uptake was about 75% in 21-day-old rats; it rapidly decreased with time when the animals were placed on a protein-free diet, approaching a level of about 1% by the 10th day of protein starvation. Over the same period plasma iEPO decreased from 55 mU/ml to 7 mU/ml. Body growth rate was 0. Following this "protein depletion period" the rats received diets containing different amounts of casein ("protein repletion period") added isocalorically to the protein-free diet to elicit a rise in body growth rate. Statistically significant relationships (p less than 0.001) were found between dietary casein concentration and body growth rate (r = 0.991), dietary casein concentration and RBC-59Fe uptake (r = 0.991), dietary casein concentration and plasma iEPO level (r = 0.992), body growth rate and RBC-59Fe (r = 0.986), and body growth rate and plasma iEPO level (r = 0.994) in hypertransfused polycythemic rats during the protein repletion period. These findings suggest that the correlation between erythropoietic activity and growth rate in the growing rat is the result of an erythropoietin-dependent operating mechanism, which appears to be independent of the ratio tissue oxygen supply/tissue oxygen demand.

Comparison of erythropoietic response to erythropoietin-secreting stimuli in mice following polycythemia induced by transfusion or hypoxia.

The erythropoietic response, measured as RBC-59Fe uptake, in response to either 24-h exposure to hypoxia or administration of dexamethasone, isoproterenol, testosterone, or erythropoietin, was determined in both posthypoxic (PH) and hypertransfused (HT) polycythemic mice. Highly significant differences between PH and HT mice exposed to hypoxia or injected with dexamethasone, isoproterenol, or testosterone were observed, isotope incorporation being always higher in PH than in HT mice. On the other hand, the response to erythropoietin did not show a significant difference between PH and HT mice. These results suggest that PH mice have been preconditioned by exposure to hypoxia in a way that makes them more sensitive to at least some kinds of erythropoietic stimuli. Since these stimuli have been shown by others to increase erythropoietin production, the results support our hypothesis that hypoxia induces sensitization of the erythropoietin-producing organ(s).

Enhanced erythropoiesis induced by hypoxia in hypertransfused, post-hypoxic mice.

The erythropoietic response, measured as RBC-59Fe uptake after 24 h-exposure to hypoxia, were determined in posthypoxic, "acute" and "chronic" hypertransfused, and posthypoxic-hypertransfused mice. Radioiron uptake by erythrocytes was about 25 times greater in post-hypoxic than in both types of hypertransfused mice. Posthypoxic-hypertransfused mice showed an erythropoietic response to hypoxia which was not significantly different from that of posthypoxic mice and about 20 times greater than those of "acute" and "chronic" hypertransfused mice. When hypertransfused mice were exposed to hypoxia before transfusions, a linear increase in 59Fe uptake values in response to hypoxia was observed with exposures between 6 and 30 h. No further increase was observed for exposures of 46 to 216 h. These results suggest a sensitization of the erythropoietin-producing organ(s) by hypoxia.

Hypoxia-induced renal and extrarenal erythropoietin production in posthypoxic or hypertransfused polycythemic rats.

The erythropoietic response, measured as 59Fe uptake by RBC, and the erythropoietin content of kidney tissue after exposure to hypobaric hypoxia were determined in both posthypoxic (PH) and hypertransfused (HT) rats. Plasma Ep titers were also measured in both PH and HT nephrectomized rats treated similarly. PH and HT rats with similar degrees of polycythemia greatly differed in their responses to hypoxia, with radioiron uptake by RBC about six times higher in the former than in the latter. Kidney Ep titers were significantly enhanced after exposure to hypoxia in either normal or polycythemic rats. However, kidney Ep titers in PH rats were significantly higher than the values found in normal or HT rats. Plasma Ep titers in both PH and HT nephrectomized rats exposed to hypoxia were similar and significantly lower than the plasma values of normal or nephrectomized, nonpolycythemic rats treated similarly. These results suggest that exposure to hypoxia induces sensitization of the renal, but not extrarenal, mechanism involved in Ep production.

Exocrine secretion of immunoreactive erythropoietin from the rat submaxillary gland.

The physiological role of immunoreactive erythropoietin (iEp) in rodent submaxillary glands (SMG) is largely unknown. We studied in vivo the effects of cholinergic and adrenergic agents in male rats with respect to exocrine secretion of iEp into saliva. Intravenous administration of metacholine (20 micrograms/kg), norepinephrine (30 micrograms/kg), and isoproterenol (30 micrograms/kg) resulted in equal volumes of saliva over 1 h. None of the drugs altered circulating plasma levels and kidney concentrations of iEp. Salivary secretions induced by either norepinephrine or isoproterenol, both adrenergic agonists, contained high levels of iEp and a significant depletion of gland content was observed, suggesting that SMG exocrine iEp secretion is mediated by adrenergic receptors. In contrast, metacholine-stimulated glands retained their full iEp content and iEp was undetectable in saliva, indicating that cholinergic activity is not associated with exocrine secretion of iEp from SMGs.

Damage of tracer erythropoietin results in erroneous estimation of concentration in mouse submaxillary gland.

It has been previously reported that 1) plasma erythropoietin (Epo) titer during exposure to hypobaria is lower in nephrectomized rats and mice whose submaxillary glands (SMG) were either ablated or atrophied than in nephrectomized controls whose SMG were intact and 2) that the gland shows one of the highest levels of immunoreactive Epo (iEpo) in the body. The latter observation, however, was questioned recently when it was observed that SMG extracts degrade labeled Epo used as tracer antigen in the radioimmunoassay (RIA), thus giving invalid estimates of Epo. Since this interpretation was in turn questioned, the present study was conducted to obtain more information on the subject and make these conflicting points clear. Investigation of the reported/possible degradation of Epo by SMG homogenates was conducted via polyacrylamide gel electrophoresis followed by radioautography or by a RIA in solid phase in which there was no simultaneous incubation of the tracer antigen with the SMG homogenates. It was observed that 125I-labeled rhEpo was degraded when incubated with SMG homogenates. Degradation was rapid, being evident when incubation lasted 30 minutes, and occurred in the presence of a protease inhibitor. It showed a high degree of specificity since it did not occur when Epo was incubated with kidney homogenate or normal mouse serum. SMG homogenate did not degrade labeled thyrotrophic hormone and degraded alpha interferon (IFN-alpha) only partially. When estimates of iEpo in SMG homogenate were performed in conditions of simultaneous (SI-RIA) or nonsimultaneous (NSI-RIA) incubation of the homogenate with tracer Epo, it was observed that while estimates of Epo in plasma were similar in both types of RIA and somewhat higher in kidney homogenate in the SI-RIA than in the NSI-RIA, estimates of Epo in SMG were about 60 times higher in the former than in the latter. Therefore, it could be concluded that most of the Epo detected by standard RIA in SMG homogenate does not represent true Epo because of damage of tracer Epo which determines loss of the integrity of the RIA system.

Striatal modulation of the jaw opening reflex.

The effect of striatal electrical and chemical conditioning stimulation (L-glutamate 80-160 nmoles/0.5 microl) on the jaw opening reflex (JOR) was studied in Sprague-Dawley male rats anesthetized with urethane. The JOR was evoked by stimulation of the tooth pulp of lower incisors. This response was suppressed by transection of the dental root, which indicates according with the bibliography, a specific activation of the pulp nerves. Three type of responses were obtained on the evoked JOR by conditioning stimulation of the striatum; being the main one the suppression of the reflex elicited by tooth pulp activation. A second type of response was an increase of the tooth-JOR amplitude. This effect was observed more frequently with glutamate stimulation rather than with electrical activation of the striatum. A third response was observed with chemical stimulation but not by electrical stimulation of the striatum. This was a triphasic response which consisted in an increase followed by an inhibition and a late increase of the tooth-JOR amplitude. A biphasic effect, an increase prior to a decrease of the JOR amplitude, was also recorded with a minor frequency. The distribution of effective sites for electrical and chemical stimulation within the striatum are mainly similar located in the rostral aspect of the nucleus, with the inhibitory sites in the middle of the nucleus and intermingled with the excitatory ones. The complex responses (tri/biphasic) were observed ventrally and caudally in the nucleus. On the basis of the results mentioned above, one could assume that the striatum is related to the modulation of the JOR evoked probably by nociceptive stimulation. However, activation of other type of fibers could not be ruled out.

Mandibular growth retardation in growing rats chronically exposed to hypobaria.

Weanling male Wistar rats aged 21 days were divided into three groups: initial control, normobaric, and hypobaric (C, N, and H, respectively). C rats were killed three days after being weaned. H rats were placed into an altitude chamber and maintained at 456 mb (6100 m) for 14 days. N rats were maintained at sea-level conditions. Body weight, body and tail lengths, and food intake were recorded every day. Animals were killed at the end of the experimental period, and four linear dimensions of the dried mandible were measured. The amount of food eaten by the H rats during the entire exposure period was 54.6% of that consumed by N ones. Body weight gain in H rats was 32.7% of that seen in N rats. Body length was 49.0% and tail length was 56.6% of normal. All mandibular dimensions were significantly reduced in H rats when compared with N rats and were, in general, in close relation with the reduction observed in skeletal growth. Only one dimension was reduced out of proportion, which indicates some deformation of the mandible. The average daily caloric intake related to metabolic body weight (body weight 0.75) of H rats was 60% of the N value. Efficiency of protein utilization for growth was not significantly different between both groups of rats. These results indicate that chronic exposure to hypobaria induces overall skeletal and mandibular growth retardation, which appears to be the result of a diminution in food intake because of decreased appetite.

The concentration of dietary casein required for normal mandibular growth in the rat.

To determine a suitable casein concentration for normal, undeformed mandibular growth, we placed weanling male rats on diets containing graded levels of casein between 0% and 30% for 19 days. Some weanlings were killed so that initial values could be established. Ten linear dimensions corresponding to the six skeletal units of the mandible were evaluated so that their growth rates at the end of the experimental period could be established. Other dimensions were also evaluated for study of the growth rate of the bone as a whole. The macroscopic growth of the mandible showed a sigmoidal relationship with dietary casein concentration, most of the measurements reaching a plateau at 20% casein. Within the skeletal units, four dimensions corresponding to the alveolar and symphyseal regions did not change with age and were not affected by the casein content of the diet. The remaining six dimensions-corresponding to condylar, coronoid, angular, and basal regions of the mandible-increased with age and were related positively to dietary casein concentration. Their growth patterns were not uniform, although all of them reached maximal values when the diet contained 20% casein. Therefore, deformation of the mandible appears to occur in rats fed diets with a casein concentration lower than 20%. It appears that a dietary casein concentration of 20% is required for normal, undeformed mandibular growth.

Stimulation of dental pulp with simultaneous recording of the jaw opening reflex in the rat.

The processing of nociceptive information in the central nervous system has been analysed in most studies by activation of peripheral nerves. However, the limitation of this method is the simultaneous activation of noxious and inocuous fibers. Nevertheless, the stimulation of the tooth pulp is believed to activate mainly nociceptive fibers which could be the method of choice. On the other hand, the response to nociceptive activation of the dental pulp is easily quantified by the amplitude of the jaw opening reflex, a nocifensive flexion withdrawal reflex. In this protocol we describe a technique to manufacture and implant electrodes in lower incisors of the rat and a method to prepare and insert stainless steel twisted bipolar electrodes to record the electromyographic activity of both digastric muscles, in response to nociceptive dental pulp stimulation. This approach was applied in the study of the analgesic effects of the rat's striatal stimulation.

Enhanced effect of increased erythrocyte production rate on plasma erythropoietin levels of mice during subsequent exposure to hypobaria.

The present study was undertaken to determine plasma EPO levels in response to acute exposure to hypobaria in transfused-polycythemic mice previously exposed to different forms of erythropoietic stimulation. The erythrocyte production rate (EPR) was stimulated by one of the following conditions: (1) discontinuous exposure to 456 mb during 4 weeks, (2) one weekly injection of phenylhydrazine (PHZ) during 4 weeks for induction of a compensated hemolytic state, and 3) three weekly injections of rHuEPO at a dose level of 1500 U/Kg body weight for 4 weeks. Treated and control mice received different volumes of packed red cells in order to obtain hematocrit values around 65% in all groups. Mice from each group were exposed to 456 mb during 14 h, 4 days after transfusion. Mice were bled immediately through cardiac puncture after removal from the hypobaric chamber. Plasma EPO titer was estimated by radioimmunoassay. Two observations were worth noting: (1) plasma iEPO level was very low in mice with transfusion polycythemia exposed to hypobaria, the value of 12 +/- 1.3 mU/ml being significantly different from that of 180 +/- 18 mU/ml found in normocythemic mice similarly exposed, and (2) plasma iEPO levels during EPRs were previously stimulated by different conditions (hypobaria, PHZ and rHuEPO) were at least as high as those found in normocythemic mices similarly exposed. All these values were significantly higher than the value corresponding to polycythemic mice whose EPRs were not stimulated before exposure to hypobaria.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunoreactive erythropoietin in rodent submaxillary gland. Autonomic control of exocrine secretion and factors influencing its concentration.

The SMG of mice and rats contain a heterologous group of biologically active factors. Some are well known, can be obtained at high purity and are well-characterized. There is strong evidence for the presence of others although they have not been purified. Finally, some of them are questionable and/or have not yet been characterized. EPO would be one of the factors whose presence in the SMG is strongly suspected, although its biological activity has not been demonstrated yet. Its presence in the gland, therefore, is only supported by radioimmunoassay data and immunocytochemical methods. Immunoreactive EPO is undetectable in the mouse SMG until the 30th day of postnatal life, increasing thereafter at a uniform rate and reaching adult levels by 50-60 days of age. The parallelism between its concentration in extracts of the gland, the size and relative proportion of GCT cells, could be accepted as indirect evidence for its localization in these cells. The rise in iEPO concentration in SMGs after androgen treatment, its fall following orchiectomy, and its reduction after duct ligation in proportion to the degree of degranulation of GCT cells lend support to the above hypothesis. Salivary secretions induced by either NE or ISO contain high levels of iEPO. A significant depletion of gland content is also observed. These two sets of data indicate that SMG exocrine iEPO secretion occurs and that this secretion is mediated by adrenergic receptors. The question whether the SMG also functions as an endocrine organ in relation to EPO can not be answered at present.

Erythropoietin formation during hypoxia in mice with impaired responsiveness to erythropoietin induced by irradiation or 5-fluorouracil injection.

Plasma erythropoietin levels during continuous exposure to hypobaric hypoxia in mice with marrow aplasia induced by whole body X-irradiation of 5-fluorouracil injection were higher than in control mice similarly exposed. There findings give support to the hypothesis that a relationship exists between erythropoietin production rate and erythroid responsiveness to the hormone.

The acidic glycosaminoglycans in gasric mucosa of athyroid rats: The effects of L-tri-iododthyronine.

Hyaluronic acid is increased about 12%, whereas dermatan sulfate is decreased about 34%, in gastric mucosa of athyroid rats when compared with controls. Administration of 2 doses of L-tri-iodothyronine reversed the effects of thyroidectomy.

The synovial fluid hyaluronic acid in rheumatoid arthritis.

The intrinsic viscosity of hyaluronic acid in synovial fluid decreases significatively in mild and severe arthritis (24% and 37% respectively). Variation in hyaluronic acid concentration parallels the above results. Chondroitin-6-sulfate can be detected in about 30% of the arthritic fluids.