Isolation and properties in culture of human adrenal capillary endothelial cells

The isolation of human adrenal capillary endothelial (HACE) cells without resort to fluorescence activated cell sorting is described, together with their properties in culture. HACE cells were isolated by plating collagenase digests at high dilution in the presence of endothelial cell growth supplement, followed by clonal selection of endothelial colonies. HACE cells exhibit a typical endothelial 'cobblestone' morphology at confluence and formed 'tubes' when seeded onto 'Matrigel'. They are positive for human MHC1, and the endothelial markers ENDOCAM (CD31) and weakly CD34, they also take up dil-acetyl low density lipoprotein but are negative for Factor VIII. Their growth is strongly stimulated by FGF and inhibited by TGF-beta I. Like their much studied bovine counterparts they are robust in culture, retaining the properties described up to senescence. HACE cells provide a readily available alternative to human umbilical vein endothelial cells in that they are easily isolated pure and in quantity. They should be particularly useful in studies where human capillary, as opposed to large vessel endothelium, is required.     

Fever and regional blood flows in wethers and parturient ewes

To determine whether the reported absence of fever in full-term-pregnant ewes might be associated with shifts of regional blood flows from thermogenic tissues to placenta during this critical period, fevers were induced twice by injections of Escherichia coli lipopolysaccharide (LPS, 0.25 microgram/kg iv) into each of six Merino ewes from 8 to 1 days prepartum, and their regional blood flow distribution was measured with radioactive, 15-microns-diam microspheres before and during the rise in fever (when their rectal temperature had risen approximately 0.4 degree C). Unexpectedly, fever always developed, rising to heights not significantly different at any time before parturition [4-8 days prepartum = 0.81 +/- 0.23 degree C (SE); 1-3 days prepartum = 0.75 +/- 0.17 degree C) and similar to those in three wethers treated similarly (0.90 +/- 0.10 degree C). Generally, during rising fever, blood flow in the ewes shifted away from heat loss tissues (e.g., skin, nose) to heat production tissues (e.g., shivering muscle, fat) and cardiac output increased; blood flow through redistribution organs (e.g., splanchnic bed) decreased. The reverse occurred during defervescence. Utero-placental blood flow remained high in the febrile ewes. These regional blood flow distributions during febrigenesis and lysis are essentially the same as those during exposures to ambient cold and heat, respectively. Some differences in the responses of cardiac output and its redistribution, however, were apparent between wethers and pregnant ewes. We conclude that 1) the previously reported "absence of fever in the full-term-pregnant sheep" should not be regarded as a general phenomenon and 2) full-term-pregnant sheep support fever production without sacrificing placental blood flow.     

THE NORMAL FINE STRUCTURE OF OPOSSUM TESTICULAR INTERSTITIAL CELLS

The interstitial tissue of the opossum testis includes interstitial or Leydig cells, macrophages, and small cells which morphologically resemble mesenchymal cells. The latter are thought to give rise to mature interstitial cells. The most prominent feature of the interstitial cell cytoplasm is an exceedingly abundant agranular endoplasmic reticulum. This reticulum is generally in the form of a meshwork of interconnected tubules about 300 to 450 A in diameter, but occasionally it assumes the form of flattened, fenestrated cisternae resembling those of pancreatic acinar cells, except for the lack of ribonucleoprotein particles on the surface of the membranes. The interstitial cells vary considerably in their cytoplasmic density. The majority are quite light, but some appear extremely dense, and in addition usually have a more irregular cell surface, with numerous small pseudopodia. These differences may well reflect variations in physiological state. Cytoplasmic structures previously interpreted as "crystalloids" consist of long bundles of minute parallel tubules, each about 180 A in diameter, which seem to be local differentiations of the endoplasmic reticulum. The mitochondria are rod-shaped, and contain a moderately complex internal membrane structure, and also occasional large inclusions that are spherical and homogeneous. The prominent juxtanuclear Golgi complex contains closely packed flattened sacs and small vesicles. The results of the present study, coupled with biochemical evidence from other laboratories, make it seem highly probable that the agranular endoplasmic reticulum is involved in the synthesis of the steroid hormones produced by the interstitial cell. This finding therefore constitutes one of the first functions of the agranular reticulum for which there is good morphological and biochemical evidence.     

A possible role for cyclic GMP in mediating the effect of luteinizing hormone releasing hormone on gonadotropin release in dispersed pituitary cells of the female rat.

In continuing studies on cyclic nucleotide involvement in the regulation of gonadotropin release, we have measured the cyclic nucleotide content and rate of LH and FSH release during stimulation by LHRH of dispersed overnight cultured cells from the pituitaries of adult female rats. The minimal effective concentration of LHRH was 0.1 nM and half maximal stimulation of gonadotropin release was observed in the presence of 1.0 nM LHRH. Significant release of both LH and FSH was detectable after only 10 min in the presence of 5 nM LHRH. The presence of fetal calf serum (FCS) in the overnight culture medium increased basal cGMP levels significantly, whereas horse serum (HS) had no effect, therefore all experiments were conducted on cells cultured in the presence of HS. Treatment of the cultured cells with the phosphodiesterase inhibitors theophylline (TH) or isobutyl-methyl-xanthine (MIX) revealed a preferential stimulatory effect of TH on basal cAMP levels and of MIX on cGMP levels. Throughout these experiments, LHRH had no effect on cAMP levels. In the presence of MIX, concentrations of the releasing hormone as low as 1 nM induced a significant rise in the level of cGMP whereas in its absence, cGMP levels appeared to be unchanged by LHRH. The increase was detectable after 10 min of incubation. MIX alone slightly increased LH and FSH release and significantly potentiated the response of the cells to increasing doses of LHRH up to, but not beyond, 10 nM. The data support the possibility that cGMP may be involved in the mechanism of action of LHRH.