Sustained microgravity reduces intrinsic wound healing and growth factor responses in the rat.

Spaceflight is known to diminish bone mass and reduce immune function, suggesting that repair of connective tissue might be impaired in a microgravity environment. Fisher 344 rats were used to test wound healing responses in the orbiting Space Shuttle Endeavour by preflight implantation of polyvinyl acetal sponge disks in which pellets were placed to release either platelet-derived growth factor (PDGF-BB), basic fibroblast growth factor (bFGF), or placebo. Control groups on the ground included a matched environment group in similar housing modules and temperature control groups in cages at 22 degreesC and 28 degreesC. After 12 days of implantation and 10 days in orbit, the removed sponges were analyzed for histological and biochemical responses. Growth factor responses were histologically evident after release of PDGF-BB and bFGF in ground controls, whereas only immediate-release bFGF and delayed-release PDGF-BB showed significant responses in microgravity. Biochemical data confirmed that cellularity was increased by both factors in ground sponges; however, this response was significantly blunted in flight sponges (P<0.005, ANOVA), irrespective timing of factor release. Collagen content was 62% lower in sponges from animals with 10 days of microgravity exposure (P<0.01, ANOVA) and further reduced by bFGF. These data suggest that orbital exposure retards the capacity of wounds to heal and respond to exogenous stimuli.

Effect of pH and ionic strength on the spectrophotometric assessment of nucleic acid purity.

The ratio of absorbance at 260 and 280 nm (the A260/280 ratio) is frequently used to assess the purity of RNA and DNA preparations. Data presented in this report demonstrate significant variability in the RNA A260/280 ratio when different sources of water were used to perform the spectrophotometric determinations. Adjusting the pH of water used for spectrophotometric analysis from approximately 5.4 to a slightly alkaline pH of 7.5-8.5 significantly increased RNA A260/280 ratios from approximately 1.5 to 2.0. Our studies revealed that changes in both the pH and ionic strength of the spectrophotometric solution influenced the A260/280 ratios. In addition, the ability to detect protein contamination was significantly improved when RNA was spectrophotometrically analyzed in an alkaline solution. UV spectral scans showed that the 260-nm RNA absorbance maximum observed in water was shifted by 2 nm to a lower wavelength when determinations were carried out in Na2HPO4 buffer at a pH of 8.5. We found RNA A260/280 ratios to be more reliable and reproducible when these spectrophotometric measurements were performed at pH 8.0-8.5 in 1-3 mM Na2HPO4 buffer.

DNAzol: a reagent for the rapid isolation of genomic DNA.

In this report, we present DNAzol, a patent-pending DNA isolation reagent containing guanidine thiocyanate and a detergent mixture. It is a complete, nontoxic and ready-to-use reagent for the isolation of genomic DNA from various biological sources. In the DNAzol protocol, a biological sample is homogenized (or lysed) in DNAzol, and the DNA is precipitated with ethanol, washed and dissolved in 8 mM NaOH. Following pH adjustment, the DNA can be used immediately for analysis or stored at 4 degrees C. The entire isolation can be completed in 20-30 min, and a wide range of DNA molecules can be isolated including genomic DNA and DNA fragments down to 0.1 kb in length. If necessary, samples can be stored in DNAzol at room temperature for extended periods of time. The isolated DNA is ready for PCR, Southern blotting and other molecular biology applications without any additional purification.

Versatile fluid-mixing device for cell and tissue microgravity research applications.

Microgravity life-science research requires hardware that can be easily adapted to a variety of experimental designs and working environments. The Biomodule is a patented, computer-controlled fluid-mixing device that can accommodate these diverse requirements. A typical shuttle payload contains eight Biomodules with a total of 64 samples, a sealed containment vessel, and a NASA refrigeration-incubation module. Each Biomodule contains eight gas-permeable Silastic T tubes that are partitioned into three fluid-filled compartments. The fluids can be mixed at any user-specified time. Multiple investigators and complex experimental designs can be easily accommodated with the hardware. During flight, the Biomodules are sealed in a vessel that provides two levels of containment (liquids and gas) and a stable, investigator-controlled experimental environment that includes regulated temperature, internal pressure, humidity, and gas composition. A cell microencapsulation methodology has also been developed to streamline launch-site sample manipulation and accelerate postflight analysis through the use of fluorescent-activated cell sorting. The Biomodule flight hardware and analytical cell encapsulation methodology are ideally suited for temporal, qualitative, or quantitative life-science investigations.

Quantitative immunoassay of total cellular GAP junction protein connexin32 during liver regeneration using antibodies specific to the COOH-terminus.

A radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA) were used to determine relative concentrations of liver connexin32 (CX32) in rats. The RIA and ELISA utilize synthetic peptides corresponding to regions of the carboxyl-terminus and antibodies raised in rabbits against these peptides. Assuming that affinities of antisera are similar for peptide and native CX32, total cellular CX32 was found to exceed the amount of gap junction protein at the cell surface calculated from morphometric analyses by 1.5-2.0 fold. This finding raises the possibility that some of the protein is present in cytoplasmic compartments or as occult precursors in the plasma membrane. Studies of CX32 content in regenerating rat liver support this conclusion and show a time course of loss and recovery of CX32 that agrees with those reported in studies using other techniques.

The biological and immunological properties of fractionated atrial extracts from young and old rats.

We have previously reported that the biological activity of rat atrial extract declines with age. The present study was undertaken to further evaluate the natriuretic, hypotensive and immunological properties of fractionated and HPLC purified atrial extracts prepared from young and old rats. Acetic acid extracts were prepared and subsequently fractionated by gel permeation chromatography. The high (greater than 10,000 daltons) and low (less than or equal to 10,000 daltons) molecular weight fractions were collected, lyophilized and assayed. Radioimmunoassay competitive binding curves of the initial and fractionated extracts were parallel (p greater than 0.05) to the synthetic ANP standard. No differences in parallelism (p greater than 0.05) were observed in the natriuretic activity of the initial extracts, the low molecular weight (LMW) fractions from both age groups, the 290 day high molecular weight (HMW) fraction or the synthetic ANP standard. However, the natriuretic activity of the 15 day HMW fraction was significantly attenuated compared to the other treatment groups (p less than 0.05). The initial 15 day extract was also significantly more hypotensive than the 290 day extract (p less than 0.05). HMW extracts were subjected to HPLC and the resulting immunoreactive ANP peak was reassayed. Based on SDS-PAGE and immuno blot analysis, the HPLC purified fraction was found to contain only immunoreactive proANP. Subsequent bioassay revealed greater hypotension and reduced natriuretic activity in the 15 day proANP fraction in comparison to a similarly prepared extract from older animals. Thus, we conclude that qualitative differences in the biological properties of atrial extracts may be ascribable to age-related changes in the composition of proANP or to other undefined biologically active atrial substance(s).

Age-related differences in the natriuretic and hypotensive properties of rat atrial extracts.

The natriuretic, hypotensive, and immunological properties of atrial extracts (AE) prepared from male and female, prepubertal, adult, and old rats were evaluated. Groups of animals were killed at ages 15, 25, 32, 39, 46, 56, and 290 days of age, and the atria were homogenized and extracted in 1.0 M acetic acid. Boiled, lyophilized extracts were stored and subsequently evaluated by bioassay and RIA. No sex differences (P greater than 0.05) were observed in the natriuretic, hypotensive, or immunoreactive properties of AE from rats of identical age. On the basis of bioassay determinations, no significant differences (P greater than 0.05) were observed in the natriuretic activity of AE prepared from rats of different ages. In contrast, AE-induced hypotension was significantly lower in bioassay rats receiving AE from 290-day-old rats than in rats infused with AE from 15-, 39-, or 56-day-old animals. Despite this reduced hypotensive effect, significant differences in RIA or bioassay parallelism were not observed between the tissue extracts and a synthetic atrial natriuretic factor (ANF) standard. Estimation of ANF content revealed that the quantity of ANF per heart as well as per microgram of DNA rose steadily with age. Two independent methods of ANF quantification (i.e. rat bioassay and RIA) yielded different estimates of atrial ANF content except at 15 days of age where the bioassay/RIA ratio was approximately 0.95. At 39, 56, and 290 days of age the ratios were 0.51, 0.44, and 0.53, respectively. A significant age-dependent decline in biological activity was noted when the hypotensive and natriuretic responses were normalized to an equivalent quantity of immunoreactive ANF. On the basis of these findings, we conclude that significant age-dependent differences exist in atrial ANF content and, more importantly, in the biological and immunological activity of rat AE. Since the composition of AE is not well defined, analysis of biological activity solely on the basis of RIA determinations may be insufficient to adequately evaluate the physiological properties associated with these extracts.

The effects of gonadotropin-releasing hormone and estradiol on luteinizing hormone biosynthesis in cultured rat anterior pituitary cells.

This study investigated the effects of physiological concentrations of GnRH and estradiol (E2) on LH biosynthesis and release using cultured anterior pituitary cells. Pituitaries from female rats were enzymatically dispersed and cultured for 48 h in steroid-free alpha-Modified Eagle's Medium, followed by a 24-h culture in medium with or without E2. The cells were then incubated for a 4-h (Exp 1 and 2) or 8-h (Exp 3) period in medium containing radiolabeled precursors with or without GnRH. Radioactive precursor incorporation into LH was determined by immunoprecipitation, while immunoreactive LH (iLH) content was quantified by RIA. In the first experiment, all concentrations of E2 (10(-11)-10(-8) M) enhanced iLH release in response to 1 nM GnRH, confirming previous reports. GnRH increased [3H]glucosamine (3H-Gln) incorporation into LH, but had no effect on [35S]methionine (35S-Met) incorporation. The higher concentrations of E2 enhanced GnRH-stimulated 3H-Gln LH production. In the second experiment, the effects of GnRH (10(-9) M) and E2 (5 X 10(-10) M) on the incorporation of [3H]galactose, [3H]mannose, [3H]fucose, or [35S]sulfate into LH were investigated. Although all precursors were incorporated into LH, no specific effect of GnRH and/or E2 on incorporation of any of the precursors into LH was noted. In Exp 3, pituitary cells were cultured with or without 0.5 nM E2 followed by an 8-h incubation with varying physiological concentrations of GnRH (10(-11)-10(-9) M) and radiolabeled precursors (3H-Gln and 35S-Met). GnRH stimulated iLH release in a dose-dependent manner, and this response was enhanced by E2. GnRH also increased the incorporation of both 3H-Gln and 35S-Met into LH, but the dose of GnRH required for this response was dependent upon the estrogen environment. In the absence of E2, only 10(-9) M GnRH increased 3H-Gln LH and 35S-Met LH production, whereas in cells exposed to E2, all concentrations of GnRH (10(-11)-10(-9) M) increased 3H-Gln LH and 35S-Met LH production. In all experiments, the specific activity of radiolabeled LH released under basal conditions was greatly reduced by stimulation with GnRH. These results suggest that GnRH regulates both LH glycosylation and LH polypeptide synthesis and that E2 lowers the physiological concentration of GnRH necessary to stimulate this biosynthetic response. Moreover, estrogen's enhancement of GnRH-stimulated LH release appears to be due to its action on mechanisms regulating the release of previously synthesized stored hormone as well as the release of newly synthesized LH.

Effects of a low calcium environment on luteinizing hormone biosynthesis in cultured rat anterior pituitary cells.

The purpose of this study was to investigate the effects of lowering the extracellular calcium concentration on GnRH-stimulated LH glycosylation and LH translation, as measured by the incorporation of [3H]glucosamine (3H-Gln) and [35S]methionine (35S-Met) into immunoprecipitable LH. Cultured anterior pituitary cells, previously exposed to estradiol (5 X 10(-10) M) to maximize precursor incorporation were incubated for 4 h in normal calcium (2.5 mM) or low calcium medium (less than 15 microM) containing radiolabeled precursors with or without 1 nM GnRH. In the presence of normal calcium, GnRH significantly increased 3H-Gln-labeled LH in the medium (278%) and cells (290%), as well as total (cells plus medium) 3H- Gln LH (280%) compared to the control value (no GnRH). GnRH also significantly increased the 35S-Met LH released into the medium (164%) and total 35S-Met LH (186%) over control values. Depletion of extracellular calcium completely inhibited GnRH-stimulated 3H-Gln LH and 35S-Met LH production. Total immunoreactive LH (iLH), as measured by RIA, was also increased significantly by GnRH treatment in the presence of calcium, but this response was prevented by removal of calcium from the medium. Lowering extracellular calcium had no effect on cellular uptake or incorporation of 3H-Gln or 35S-Met into total trichloroacetic acid-precipitable protein. Approximately 80% of newly synthesized LH was released into the medium in all treatment groups independent of whether calcium or GnRH was present. The specific activity (disintegrations per min/microgram iLH) of radiolabeled LH released into the medium was significantly reduced by treatment with GnRH due to the large amount of unlabeled iLH released into the medium. However, when the cells were incubated in low calcium, the SA of 3H-Gln LH and 35S-Met LH in the medium was unaltered by GnRH, whereas GnRH-stimulated iLH release was inhibited. We conclude that GnRH stimulation of LH glycosylation and LH apoprotein synthesis involves extracellular calcium-dependent events, and the release of newly synthesized LH is closely coupled to LH biosynthesis and is less dependent on extracellular calcium, whereas the GnRH-stimulated release of previously synthesized, stored LH is dependent on extracellular calcium.

Dissociation and enrichment of rat atrial myocytes containing atrial natriuretic factor (ANF).

Methodologies developed for the dissociation and subsequent enrichment of muscle and nonmuscle cells from atrial myocardium were used to evaluate the contribution of these cell populations to the natriuretic, diuretic and vasoactive properties of crude atrial tissue extracts. Suspensions of single cells, which contained approximately 34% myocytes, were prepared from atrial tissue blocks with a collagenase-trypsin digestion followed by gentle mechanical disruption. Differential centrifugation and unit gravity sedimentation techniques were employed to enrich the 'muscle' and 'nonmuscle' cell suspensions to a purity of approximately 91 and 95%, respectively. Cell extracts were bioassayed for natriuretic activity in saline-expanded, pentobarbital-anesthetized, female rats. Extracts obtained from 'initial' and 'muscle' cell suspensions significantly enhanced sodium and chloride excretion as well as urine flow while extracts from 'nonmuscle' cell suspensions had no effect on renal function. Sodium excretion was dose-dependent and increased linearly with increasing numbers of extracted and infused myocytes. This simple two-step centrifugation and sedimentation protocol can be utilized to obtain enriched atrial myocyte populations for subsequent physiologic and biochemical studies.

Electrophoretic separation of kidney and pituitary cells on STS-8.

A Continuous Flow Electrophoresis System (CFES) was used on Space Shuttle flight STS-8 to separate specific secretory cells from suspensions of cultured primary human embryonic kidney cells and rat pituitary cells. The objectives were to isolate the subfractions of kidney cells that produce the largest amounts of urokinase (plasminogen activator), and to isolate the subfractions of rat pituitary cells that secrete growth hormone, prolactin, and other hormones. Kidney cells were separated into more than 32 fractions in each of two electrophoretic runs. Electrophoretic mobility distributions in flight experiments were spread more than the ground controls. Multiple assay methods confirmed that all cultured kidney cell fractions produced some urokinase, and five to six fractions produced significantly more urokinase than the other fractions. Several fractions also produced tissue plasminogen activator. The pituitary cells were separated into 48 fractions in each of the two electrophoretic runs, and the amounts of growth hormone (GH) and prolactin (PRL) released into the medium for each cell fraction were determined. Cell fractions were grouped into eight mobility classes and immunocytochemically assayed for the presence of GH, PRL, ACTH, LH, TSH, and FSH. The patterns of hormone distribution indicate that the specialized cells producing GH and PRL are isolatable due to the differences in electrophoretic mobilities.

An in vitro model for studies of intercellular communication in cultured rat anterior pituitary cells.

The formation of intimate associations among different hormone-secreting cells within the rat adenohypophysis may serve as a possible site for physiologic regulation. In this report we describe a high density plating method which enables us to study cell-to-cell interactions within anterior pituitary cell cultures. Trypsin-dispersed pituitary cell suspensions attach rapidly (within 6 hr) and quantitatively (95-97%) to glass or plastic surfaces when plated in medium containing microM calcium concentrations (pH 7.6-7.8). Freshly plated cell suspensions obtained from female pituitary glands contained subpopulations of mammotrophs 49.3%, somatotrophs 30.3%, gonadotrophs 12.6%, corticotrophs 3.4% and thyrotrophs 1.5%. Epithelial cell colonies were formed during a 3-day culture period as the cells flattened and re-established contacts with neighboring cells. Freeze-fracture electron microscopic analysis of these colonies produced morphological evidence for direct intercellular contacts among the hormone-secreting cells. Large areas of tight junctions and small gap junctions were identified on the membranes of the epithelial cells within these colonies. Cells which contained tight junctions usually contained microvilli and morphological signs of active hormone secretion. Small junctional plaques containing tightly packed intramembrane particles were also occasionally found on the membranes of cells which were actively secreting pituitary hormones. The high density plating procedure which is described in this report provides greater opportunity for cell-cell interaction and thus may prove to be a useful model for evaluating the role of intercellular communication within this tissue.

Characterization of the luteinizing hormone response to continuous infusions of gonadotropin releasing hormone using perifused pituitaries from intact, ovariectomized and steroid-treated rats.

Anterior pituitary glands obtained from rats at various stages of the estrous cycle and from short-term ovariectomized (OVX) rats with or without physiological replacement of estradiol-17 beta (E2) and/or progesterone (P), were perifused in vitro with continuous infusions (4 h) of gonadotropin releasing hormone (GnRH) (12 ng/h). In all treatment groups the in vitro pattern of luteinizing hormone (LH) release in response to GnRH was characterized by an initial low rate of LH release (initial phase; 20-70 min) followed by a significant augmented rate of secretion during the late phase (120-240 min) with the exception of the estrous and OVX + P groups in which LH was released at a constant rate. The total amount of LH released in response to GnRH was similar for glands removed on estrus, diestrus-I (D-I) and D-II, but increased significantly for glands removed on 0900 h proestrus with a further increase at 1400 h proestrus. Ovariectomy reduced the total LH released in vitro by 50-60% and 85-90% compared with estrous, D-I, D-II and proestrous groups, respectively. In vivo treatment of OVX rats with E2 restored the in vitro LH response to levels comparable to those in the 0900 h proestrous group while treatment with P + E2 further increased the rate of LH release in the initial phase to levels similar to those observed in the 1400 h proestrous group. In all treatment groups, addition of 5 microM cycloheximide to the perifusion media significantly inhibited GnRH-stimulated LH release during the late phase without significantly altering the initial LH response except in the OVX + E2P group in which it was partially inhibited. These results demonstrate that perifused pituitaries maintain their characteristic responsiveness to GnRH in vitro. Furthermore, they indicate that LH secretion in response to continual GnRH stimulation involves protein synthesis-independent and -dependent components. E2, in vivo, enhances the magnitude of both components in response to GnRH in vitro, while E2 + P may further enhance the initial in vitro response to GnRH through a protein synthesis-dependent mechanism.

Fluorometric quantification of DNA in cells and tissue.

The validation of a simple and rapid DNA solubilization procedure is described. Quantitative extraction of intact, polymerized DNA was achieved by cell lysis or tissue homogenization in an ammonium hydroxide-Triton X-100 solution. The solubilization procedure inactivates endogenous DNAase and increases the fluorescence-enhancement activity of the extracted DNA, thereby eliminating the need for enzyme treatment or exposure to high salt solutions. The extracts can be utilized directly in a sensitive fluorescence-enhancement assay with bisbenzimidazole (Hoechst 33258) reagent. Estimates of DNA cell content were unaffected by the number of cells lysed or the volume of lysate employed in the assay. In all cases, the solubilized DNA estimates were linear and parallel to the bovine DNA standard. The optimum range for estimation of DNA in this assay is 5-150 ng. In addition, estimates of DNA obtained with this method and the standard diphenylamine assay were in excellent agreement. This simple, one-step DNA extraction procedure can be utilized in conjunction with Hoechst reagent to obtain quantitative estimates of DNA levels in cell or tissue extracts.

Culture of human pituitary prolactin and growth hormone cells.

Fragments of pituitary tissue obtained from a total of 37 patients with either breast cancer, diabetic retinopathy, galactorrhea, or acromegaly were dissociated into single cell suspensions prior to cell culture. Release of human growth hormone (hGH) and human prolactin (hPRL) into the culture medium was measured by radioimmunoassay. During a 3-week culture period, prolactin cells released 9--13 times the intracellular levels of hPRL at the time of seeding, whereas hGH release from growth hormone cells was only 1--2 times that of their initial intracellular level during this same time. Both growth hormone and prolactin cells retained distinctive ultrastructural features during culture. The prolactin cells responded to TRH stimulation by elevated release of PRL into the medium. No evidence for mitotic division of prolactin cells in vitro was found.

Characterization of mammotrophs separated from the human pituitary gland.

Human pituitary tissues from 27 patients and 7 persons post mortem were dissociated into single cell suspensions. On the average, 23% of the cells were mammotrophs. The concentration of prolactin in these suspensions averaged 3.8 ng/1,000 cells. After cell separation by velocity sedimentation at unit gravity, mammotrophs and other cell types were enriched twofold to threefold. The separated mammotrophs retained structural integrity at light and electron microscopic levels. In eight separation experiments, cells recovered from different gradient regions were assayed for intracellular prolactin levels. In cells from "normal" subjects, 8.5% of the prolactin recovered from the gradient was associated with large mammotrophs, whereas in patients with breast cancer, 28% of the hormone was associated with large mammotrophs. The number of mammotrophs recovered from this gradient region (beyond fraction 6) was doubled in breast cancer (2 expts). These mammotrophs showed areas of hypertrophied Golgi and endoplasmic reticulum. Culture of the separated cells from 1 patients with diabetes and 2 patients with breast cancer for 21 days showed that mammotrophs in the upper gradient fractions (diabetic) secreted seven times more hormone than those in the lower regions, whereas those mammotrophs from patients with breast cancer that fell to the lower gradient regions secreted 15 times more prolactin than did those in the upper regions. These data suggest that pituitaries of patients with breast cancer contain a small pool (10-20%) of hypertrophied mammotrophs that have the potential for significant secretory activity in vitro.

Maintenance of separated rat pituitary mammotrophs in cell culture.

Dispersed female rat anterior pituitary cells were cultured in Medium 199 containing 20% fetal calf serum for 30 days. Prolactin levels in the culture medium remained relatively constant during this time, ranging from 30-40 ng/10,000 cells seeded/day. The total quantity of prolactin released into the medium was 10-15 times that originally contained in the cells. Morphological integrity of the mammotrophs was maintained. Using velocity sedimentation at unit gravity, cells from untreated, overiectomized or estrogen-primed animals were separted into several fractions, and subsequently cultured for 14 days. Not all mammotrophs secreted the same quantity of hormone during this time. The data suggest that the pituitary of the female rat is composed of a heterogeneous population of mammotrophs, and that their capacity to secrete prolactin in vitro may, in part, be reflected by the previous physiological status of the animal.