On the use of positron counting for radio-Assay in nuclear pharmaceutical production.

Current techniques for the measurement of radioactivity at various points during PET radiopharmaceutical production and R&D are based on the detection of the annihilation gamma rays from the radionuclide in the labelled compound. The detection systems to measure these gamma rays are usually variations of NaI or CsF scintillation based systems requiring costly and heavy lead shielding to reduce background noise. These detectors inherently suffer from low detection efficiency, high background noise and very poor linearity. They are also unable to provide any reasonably useful position information. A novel positron counting technique is proposed for the radioactivity assay during radiopharmaceutical manufacturing that overcomes these limitations. Detection of positrons instead of gammas offers an unprecedented level of position resolution of the radiation source (down to sub-mm) thanks to the nature of the positron interaction with matter. Counting capability instead of charge integration in the detector brings the sensitivity down to the statistical limits at the same time as offering very high dynamic range and linearity from zero to any arbitrarily high activity. This paper reports on a quantitative comparison between conventional detector systems and the proposed positron counting detector.

Automated production at the curie level of no-carrier-added 6-[(18)F]fluoro-L-dopa and 2-[(18)F]fluoro-L-tyrosine on a FASTlab synthesizer.

An efficient, fully automated, enantioselective multi-step synthesis of no-carrier-added (nca) 6-[(18)F]fluoro-L-dopa ([(18)F]FDOPA) and 2-[(18)F]fluoro-L-tyrosine ([(18)F]FTYR) on a GE FASTlab synthesizer in conjunction with an additional high- performance liquid chromatography (HPLC) purification has been developed. A PTC (phase-transfer catalyst) strategy was used to synthesize these two important radiopharmaceuticals. According to recent chemistry improvements, automation of the whole process was implemented in a commercially available GE FASTlab module, with slight hardware modification using single use cassettes and stand-alone HPLC. [(18)F]FDOPA and [(18)F]FTYR were produced in 36.3 ± 3.0% (n = 8) and 50.5 ± 2.7% (n = 10) FASTlab radiochemical yield (decay corrected). The automated radiosynthesis on the FASTlab module requires about 52 min. Total synthesis time including HPLC purification and formulation was about 62 min. Enantiomeric excesses for these two aromatic amino acids were always >95%, and the specific activity of was >740 GBq/µmol. This automated synthesis provides high amount of [(18)F]FDOPA and [(18)F]FTYR (>37 GBq end of synthesis (EOS)). The process, fully adaptable for reliable production across multiple PET sites, could be readily implemented into a clinical good manufacturing process (GMP) environment.

Biodistribution and Radiation Dosimetry for the Novel SV2A Radiotracer [(18)F]UCB-H: First-in-Human Study.

PURPOSE: [(18)F]UCB-H is a novel radiotracer with a high affinity for synaptic vesicle glycoprotein 2A (SV2A), a protein expressed in synaptic vesicles. SV2A is the binding site of levetiracetam, a "first-in-class" antiepileptic drug with a distinct but still poorly understood mechanism of action. The objective of this study was to determine the biodistribution and radiation dosimetry of [(18)F]UCB-H in a human clinical trial and to establish injection limits according to biomedical research guidelines. Additionally, the clinical radiation dosimetry results were compared to estimations in previously published preclinical data. PROCEDURES: Dynamic whole body positron emission tomography/X-ray computed tomography (PET/CT) imaging was performed over approximately 110 min on five healthy male volunteers after injection of 144.5 ± 7.1 MBq (range, 139.1-156.5 MBq) of [(18)F]UCB-H. Major organs were delineated on CT images, and time-activity curves were obtained from co-registered dynamic PET emission scans. The bladder could only be delineated on PET images. Time-integrated activity coefficients were calculated as area under the curve using trapezoidal numerical integration. Urinary excretion data based on PET activities including voiding was also simulated using the dynamic bladder module of OLINDA/EXM. The radiation dosimetry was calculated using OLINDA/EXM. RESULTS: The effective dose to the OLINDA/EXM 70-kg standard male was 1.54 × 10(-2) ± 6.84 × 10(-4) millisieverts (mSv)/MBq, with urinary bladder wall, gallbladder wall, and the liver receiving the highest absorbed dose. The brain, the tracer's main organ of interest, received an absorbed dose of 1.89 × 10(-2) ± 2.32 × 10(-3) mGy/MBq. CONCLUSIONS: This first human dosimetry study of [(18)F]UCB-H indicated that the tracer shows similar radiation burdens to widely used common clinical tracers. Single injections of at maximum 672 MBq for US practice and 649 MBq for European practice keep radiation exposure below recommended limits. Recently published preclinical dosimetry data extrapolated from mice provided satisfactory prediction of total body and effective dose but showed significant differences in organ absorbed doses compared to human data.

Lap Band adjustable gastric banding system: the Italian experience with 1863 patients operated on 6 years.

BACKGROUND: The Lap Band system procedure is currently the most common bariatric surgical procedure worldwide. This is an interim report of the experience of the 27 Italian centers participating in the national collaborative study group for Lap Band (GILB). METHODS: An electronic database was specifically created. It was mailed and e-mailed to all of the surgeons now performing the laparoscopic gastric banding operation in Italy. RESULTS: Beginning in January 1996, 1893 patients were recruited for the study. There were 1534 women and 359 men with a mean body mass index (BMI) of (range 30.4-83.6) and a mean age of 37.8 +/- 10.9 years (range; 17-74). The mortality rate has been 0.53% (n = 10), mainly due to cardiovascular complications (myocardial infarction, pulmonary embolism). The laparotomic conversion rate has been 3.1% (59/1893) and was higher in superobese patients (BMI>50) than in to morbidly obese patients (BMI <50) (p <0.05). Postoperative complications occurred in 193 patients (10.2%), including tube port failure (n = 79; 40.9%), gastric pouch dilation (GPD) (n = 93; 48.9%), and gastric erosion (n = 21, 10.8%). Most GPD (65.5%) occurred during the first 50 patients treated at each center. The incidence of GPD decreased as the surgeons acquired more experience. Surgery for complications was often performed by laparoscopic access, rarely via laparotomy. No death was recorded as a consequence of surgery to treat complications. Weight loss has been evaluated at the following intervals: 6, 12, 24, 36, 48, 60, and 72 months, with BMI 37.9, 33.7, 34.8, 34.1, 32.7, 34.8, and 32. CONCLUSIONS: The Lap Band system procedure has a very low mortality rate and a low morbidity rate and it yields satisfactory weight loss. Surgery for complications can be performed safely via laparoscopic access.

Laparoscopic Italian experience with the Lap-Band.

BACKGROUND: An increasing number of surgeons with different levels of experience with laparoscopic surgery and open obesity surgery have started to perform laparoscopic implantation of the Lap-Band. METHODS: An electronic patient data sheet was created and was mailed and e-mailed to all surgeons performing laparoscopic adjustable silicone gastric banding (LASGB) in Italy. Patients were recruited since January 1996. Data on 1,265 Lap-Band System operated patients (258 M/1,007 F; mean BMI 44.1, range 27.0-78.1; mean age 38, range 17-74 years) were collected from 23 surgeons performing this operation. RESULTS: Intra-operative mortality was absent. Post-operative mortality was 0.55% (7 patients) for causes not specifically related to LASGB implantation. The laparotomic conversion rate was 1.7% (22 patients). LASGB related complications occurred in 143 patients (11.3%). Pouch dilatation was diagnosed in 65 (5.2%), and 28 (2.2%) of these underwent re-operation. Band erosion was observed in 24 patients (1.9%). Port or connecting tube-port complications occurred in 54 patients (4.2%), 12 of whom required revision under general anesthesia. Follow-up was obtained at 6, 12, 18, 24, 36 and 48 months, and mean BMI was respectively 38.4, 35.1, 33.1, 30.2, 32.1 and 31.5. The percentage of patients observed at each follow-up was > 60%. There was no intra-operative mortality and no complication-related mortality, with acceptable weight loss. CONCLUSION: The LASGB operation is safe and effective, and deserves wider use for treatment of morbid obesity.

Selective expression of c-mas proto-oncogene in rat cerebral endothelial cells.

Northern blot analysis of cultured endothelial cells derived from rat cerebral resistance vessels demonstrated the presence of c-mas mRNA. This is the first description of mas expression in non-neuronal cells. c-mas message was not detectable in cultured endothelial cells derived from other vascular beds, including rat aorta and mesenteric resistance vessels. Since c-mas has been purported to regulate the proliferation response to angiotensin, the growth properties of all three endothelial cell cultures were examined. The data indicated no differences in either basal or angiotensin-stimulated cell growth among brain, mesenteric or aortic-derived endothelial cells. These data suggest that c-mas is selectively expressed in brain endothelial cells and that this proto-oncogene does not regulate cell proliferation in this cell type.

Growth properties and receptor expression in vascular smooth muscle cells from hypertensive rats.

The objective of this study was to evaluate the growth properties and receptor expression in aorta-ring derived smooth muscle cells (SMCs) cultured from control (WKY) and spontaneously hypertensive rats (SHR). SHR-SMCs exhibited a 3-4 day lag period before migrating. In addition, SHR-SMCs had a significantly higher growth rate, shorter population doubling time and higher saturation density level characteristics that were retained at higher passage levels. beta-adrenergic and angiotensin (All) receptors were measured using iodocyanopindolol (ICYP) and [3H]-All, respectively. All receptor expression was similar in both WKY and SHR-SMC cultures. WKY-SMCs exhibited little ICYP binding (Bmax 8.27 +/- 2.0 fmol/mg) while SHR-SMC binding capacity was 8 fold higher (Bmax 65 +/- 9.2 fmol/mg). In addition, the responsiveness of the beta-receptor, as assessed by adenylyl cyclase stimulation, was similar for WKY and SHR-SMCs. These data suggest that factors regulating SMC receptor expression in vitro are selective since All and adrenergic receptor densities exhibit different responses to hypertension.

Isolation and characterization of cerebral resistance vessel endothelium in culture.

Organ-derived endothelia have been shown to exhibit distinct patterns of morphology and growth responsiveness in vitro. This report describes the development, cloning and establishment of long-term serial cultures of rat vascular endothelial cells derived from cerebrocortical resistance vessels (small arteries and arterioles). Modification of our previous published technique for establishing resistance vessel-derived smooth muscle cells (RV-SMC) resulted in enhanced levels of endothelial outgrowth from collagenase-treated microvessel fragments. Although primary culture growth consisted predominantly of SMC, subsequent subcultivation of these cultures revealed the presence of distinct endothelial cell clusters within the SMC monolayer. Serial cloning of these isolates resulted in a homogeneous population of cells with the characteristic endothelial cobblestone growth pattern and positive immunofluorescence for factor VIII-related antigen. Previously established RV-SMC frozen stocks provided an additional source for obtaining resistance vessel endothelial cells. This was made possible by the slow proliferation rate of early-passage RV-SMC and their inability to withstand freezing procedures. Endothelial cells from both preparations were identical and designated resistance vessel derived endothelial cells RV-EC. Upon long-term cultivation (> P15), confluent RV-EC cultures expressed spontaneous multicellular cord development that stained positive for factor VIII-related antigen. Cell growth studies demonstrated that RV-EC were capable of significant growth when maintained in serum-free conditions. Growth kinetics using serum-free conditioned medium demonstrated mitogenic activity indicating the presence of an autocrine growth factor. Increase growth responsiveness was also noted in RV-EC when treated with a variety of peptide growth factors. These results indicate that resistance vessel endothelium can be successfully isolated and maintained in long-term serial cultures. Furthermore, the availability of cultured EC and SMC from this unique microvascular site will enable examination of cerebrovascular endothelial-smooth muscle cell interactions in vitro and may help to elucidate the mechanisms of altered vascular function in disease states.

Transcriptional regulation of calcium-activated neutral protease in cardiomyocytes of hypertensive rats.

Increased enzymatic activity of calcium-activated neutral protease II (CANP II) has been reported previously in cardiac tissues of rats with 2 kidney, 1 clip Goldblatt hypertension (2K, 1C-HT); this was associated with elevated intracellular free Ca(++). Because it was suggested that increased levels of the enzyme were responsible for the enhanced activity, CANP II mRNA expression was assessed in cardiomyocytes isolated from 2K, 1C-HT rats and from a genetic model of hypertension, the spontaneously hypertensive rat (SHR). Utilizing a rabbit probe for the large subunit of CANP II (pLM 1006 cDNA), a 3.7 kilobase mRNA band was visualized in Northern blots of poly A+ RNA. Densitometric analysis of the blots revealed that there was a significant (p < 0.005) increase in the levels of CANP II large subunit mRNA in cardiomyocytes of 2K, 1C-HT rats when compared with controls. Interestingly, CANP II mRNA levels were comparable in cardiomyocytes of SHR and Wistar Kyoto (WKY) rats. Results of nuclear runoff transcription assays indicated that enhanced expression of CANP II mRNA in 2K, 1C-HT rat hearts was regulated at the transcriptional level. The data support specific CANP II gene activation in the hearts of renal hypertensive rats.

Angiotensin II and atrial natriuretic factor receptor interactions at the blood-brain barrier.

Data from several laboratories indicate that cerebral endothelial cells possess cell surface receptors for numerous vasoactive agents including angiotensin II (AII) and atrial natriuretic factor (ANF). The intracellular messengers of these receptors as well as possible receptor interactions were explored. ANF increased cGMP 10-fold over basal levels while incubation of the microvessels with AII did not significantly affect the level of this nucleotide. In contrast, AII significantly potentiated the increase in cGMP by ANF. Incubation of cerebral microvessels with AII resulted in a significant increase in the intracellular mediator of PI hydrolysis, 1,2-diacylglycerol (DG). ANF had no affect on DG or on the AII mediated increase of DG. Finally, data at the level of receptor binding indicated that while ANF decreased [3H]angiotensin binding to cerebral microvessels, AII had no effect on the binding of ANF to its receptor. The results of the present study demonstrate that AII can potentiate the regulation of cGMP by ANF and suggest the possibility of receptor interactions in control of blood-brain barrier function.

Influx of neutrophils into the walls of large epicardial coronary arteries in response to ischemia/reperfusion.

BACKGROUND: There are several clinical situations in which large epicardial coronary arteries are deprived of blood flow, such as occurs when an obstructing thrombus or embolus lodges within a vessel or during coronary dissection. There is little information concerning the effect of flow deprivation on large epicardial coronary arteries. METHODS AND RESULTS: We studied a model in which a segment of a large epicardial coronary artery was deprived of blood flow using both proximal and distal clamps for 3 hours followed by reperfusion. On examination by light microscopy of cross sections of the arteries, 19 +/- 6 neutrophils were present in the intima of ischemic/reperfused vessels, whereas only a mean of 4 +/- 3 (SEM) were present in the intima of nonischemic vessels (p less than 0.02). On average, there were 17 +/- 9 neutrophils just under the elastic lamina in ischemic/reperfused vessels versus none in the nonischemic vessels (p less than 0.05); there were 16 +/- 10 neutrophils present within the media of ischemic/reperfused vessels, and none (p less than 0.05) in the nonischemic vessels. Electron microscopic analysis revealed that neutrophils in the ischemic/reperfused vessels were often "sandwiched" between the endothelial cells and the elastic lamina. Ultrastructural abnormalities within the myocardium also revealed damage to the microvasculature, including the presence of neutrophils within the vessels and erythrocyte stasis. To rule out the possibility that findings in the large epicardial arteries were due to toxic substances from static blood within the isolated arterial segment, a protocol was performed in which blood was removed from the isolated segment. Again, neutrophil infiltration into the vessel was observed. Resting mean epicardial coronary artery blood flow before coronary occlusion was 19 +/- 3 ml/min; mean coronary blood flow 2.5 hours after reperfusion was identical at 19 +/- 3 ml/min. Response to both endothelial-dependent vasodilation (acetylcholine) and endothelial-independent vasodilation (nitroglycerin) challenges was normal early after reperfusion but was depressed late after reperfusion, suggesting progressive vascular dysfunction and hence a form of vascular reperfusion injury in this model. CONCLUSIONS: When large epicardial coronary arteries are deprived of blood flow, followed by reperfusion in this model, neutrophils migrate into the vessel wall as well as into the microvasculature. These abnormalities are associated with reduced endothelial-dependent and endothelial-independent coronary vasodilator reserve.

Calcium and the impairment of contractions to norepinephrine in aorta isolated from the spontaneously hypertensive rat.

Helical strip contractility from hypertensive (SHR) and normotensive (WKY) rat aortas assessed in the presence of varying calcium concentrations indicated that SHR strips exhibit a higher intrinsic myogenic tone and contract less to norepinephrine (NE) in a physiological calcium concentration compared to controls. Relatively higher isometric tension was developed in the SHR in low calcium (0-0. 27 mM). While control responses were blunted by LaCl3, EGTA, and nifedipine, the SHR strips were unaffected. Addition of procaine significantly enhanced SHR contractility to NE with no effect on control strips. These data suggest that abnormal cytosolic calcium provokes an increase in myogenic tone and an impaired contractile response of aortic smooth muscle cells to NE.

Autonomic receptor interactions in isolated cardiac myocytes from hypertensive rats.

Isolated ventricular myocytes from adult (16 to 20 weeks) spontaneously hypertensive (SHR) and normotensive (WKY) rats were utilized to examine adrenergic and cholinergic receptor expression and interaction. Binding assays were performed using quinuclidinyl benzilate (QNB) and iodocyanopindolol (ICYP) for cholinergic and beta-adrenergic receptors, respectively. In addition, cAMP was measured as an index of adrenergic-cholinergic control of adenylate cyclase. Data from radioligand binding experiments indicated that muscarinic cholinergic receptors were depressed (22%) in SHR myocytes, while beta-adrenergic receptor density was comparable to that of WKY myocytes. Heterologous receptor modulation in isolated myocytes as assessed by displacement analysis with and without guanosine 5'-triphosphate (GTP), showed that carbachol displacement of QNB was shifted five fold to the right in the presence of GTP and that the beta-adrenergic agonist isoproterenol did not prevent the GTP-mediated binding alteration. In contrast, carbachol modulated the GTP-shift of ICYP displacement by isoproterenol and these effects were comparable in both WKY and SHR myocytes. Furthermore, the ability of carbachol to blunt the stimulation of adenylate cyclase by isoproterenol was also comparable in myocytes isolated from adult SHR and control animals. Thus, the observed decrement in muscarinic cholinergic receptor expression did not alter adrenergic-cholinergic interactions as assessed by displacement assays using guanine nucleotides, or the control of cAMP levels. In addition, isolated myocytes provide a useful system for analyzing receptor expression and regulation and how these parameters may be altered in the hypertensive heart.

Angiogenesis in rat aorta ring explant cultures.

This report describes the utilization of rat aorta "rings" as explants to study angiogenesis. Under short-term culture conditions, ring explants transferred at 3- to 4-day intervals showed growth (differential migration) of endothelial and/or smooth muscle cell populations. On the other hand, explants maintained for long-term (up to 3 weeks) without transfer, developed extensive out-growths of new vascular channels radiating on and through the established cell sheet. Examination of adventitia stripped ring segments, before culture revealed the presence of areas containing small vessel remnants embedded in the outer aorta wall. In addition, cultures of adventitial tissue alone yielded a variety of cell types including the occasional presence of vascular cells with developing channel formation. Immunofluorescent and ultrastructural examination of long-term aorta explant cultures revealed that these channels consisted of endothelial cells that exhibited positive reactivity for factor VIII antibody. In order to determine the influence of aorta-derived cells on vascular channel development, various vessel treatments and manipulations were performed before explant culture, to eliminate the influence of either the endothelium and/or adventitia-associated microvasculature remnants. Thus results clearly showed that the presence of both constituents is required to initiate the angiogenic response in this in vitro culture model. In addition, short-term aortic ring explant culture is an efficient and reliable technique for the isolation and culture of endothelium and smooth muscle cells from large vessels of the rat. More importantly, long-term maintenance of aorta explant cultures provides a novel self-contained system to study new vessel growth and development, and represents an attractive alternative model for defining the cellular processes regulating angiogenesis.

Cerebrovascular angiotensin II receptors in spontaneously hypertensive rats.

The objective of this study was to characterize angiotensin II (AII) receptors in cerebral capillary endothelium and to examine whether the first step in AII responsiveness, namely AII receptor binding, is aberrant in cerebral microvessels obtained from adult spontaneously hypertensive rats (SHR). The binding of [3H]angiotensin II to isolated cerebrocortical microvessels from Sprague-Dawley, Wistar-Kyoto, and SHR rats was used to characterize AII receptors on these vessels. Kinetic experiments yielded an equilibrium-derived Kd (dissociation rate constant/association rate constant) very close to that obtained from Scatchard analysis of saturation binding data. The data indicated that the two normotensive control strains exhibited comparable AII receptor affinity and binding capacity. In contrast, experiments with microvessels from adult SHR indicated a significantly higher Bmax for AII receptors relative to controls. Although experiments assessing functional endothelial alterations in the SHR to AII remain to be performed, the increase in AII receptor number suggests that an abnormality in vascular AII responsiveness may play an important role in this model of hypertension.

Autonomic receptor characterization in cultured adult rat cardiac myocytes: morphologic-functional correlation.

Adult calcium tolerant rat ventricular myocytes were maintained under serum-free culture conditions for five days. beta-adrenergic and muscarinic cholinergic receptor expression was assessed by radioligand binding determinations using 125I-iodocyanopindolol (ICYP) and [3H]-quinuclidinyl benzilate (QNB), respectively. The binding data were correlated with myocyte structural integrity and contractile responsiveness to norepinephrine (NE). During the 5 days in primary culture, beta-adrenergic and muscarinic cholinergic receptor binding capacity diminished Bmax = 17.1 to 9.2 fmol/mg protein and Bmax = 169.0 to 26.6 fmol/mg protein, respectively. The affinity of both autonomic receptors was unaltered during the period of observation. The majority of isolated myocytes were viable (65 to 85%) and remained rod-shaped for 5 days as assessed by phase contrast microscopy. Up to 2 days in vitro the rod-shaped myocytes appeared ultrastructurally similar to their in vivo counterparts and displayed intact nuclei and the usual complement of cellular organelles. From day 3, phase contrast as well as transmission electron microscopy revealed a progressive increase in autophagic vacuoles consisting primarily of disrupted mitochondria. The number of myocytes that contracted in response to norepinephrine (NE) decreased from 57.2 to 2.3% by day 5. These data indicate that adult rat cardiac myocytes maintained in serum free culture for 5 days, express beta-adrenergic and muscarinic cholinergic receptors. There is a rapid decline (50%) in muscarinic cholinergic receptor number and contractile response to NE by day 2. However, the decrease in beta-receptor Bmax by day two is insufficient to explain the severe loss of cell responsiveness to NE. This functional loss may be related, at least in part, to the ultrastructural abnormalities that are first evident at day 2 in culture. Thus, short-term myocyte cultures that retain phenotypic and physiologic characteristics of in vivo cardiac myocytes could provide a useful in vitro system for exploring pharmacologic-functional interactions in the myocardium.

Structural analysis of the calcium channel by photoaffinity labelling and limited proteolysis.

The L-type calcium channel of rabbit skeletal muscle triads, purified from digitonin extracts, was photolabelled with the dihydropyridine (+)[3H]PN 200-110. This photolabelled form was then subjected to limited proteolysis with papain and staphylococcus V-8 protease and analyzed by polyacrylamide gel electrophoresis. In the absence of proteolysis, the photolabelled channel was represented by a single protein with an apparent molecular weight of 160 kDa in the presence or absence of reducing agents. Following proteolysis, numerous photoadducts were observed with smaller molecular weights. The V-8 protease digestion pattern indicated that photoinsertion occurred in at least two distinct domains of 33 and 28 kDa. Papain digests were more extensive, generating smaller fragments of 28 and ca. 10 kDa. The results suggest that at least two distinct regions of the calcium channel interface at or near the dihydropyridine binding site, and that the binding site for these calcium antagonists resides within the channel proper, thereby modulating calcium influx.

Altered calcium regulation in the cardiac plasma membrane in experimental renal hypertension.

The factors regulating calcium homeostasis in the cardiac plasma membrane of renal hypertension in the rat (two kidney-one clip, Goldblatt model) have been studied. Comparison of the cardiac sarcolemma from control (C) and hypertensive (H) rats indicates similar protein yield and purity. Study of longer term hypertension (4 to 12 weeks) shows a decrease in the number of calcium channel receptor binding sites (Bmax C: 549 +/- 122 fmol/mg; H: 334 +/- 74 fmol/mg) as well as a depressed calcium pumping ATPase activity (C: 7.6 +/- 2.5 nmol/mg/min; H: 3.8 +/- 1.5 nmol/mg/min). Furthermore, there is a decreased rate of Na+-Ca2+ exchange (C: 5.4 +/- 1.9 nmol/mg/5 s; H: 2.3 +/- 0.9 nmol/mg/5 s). Study of short-term hypertension (1 to 4 weeks) indicates that the earliest change occurs at 1 week with decreased calcium pumping ATPase due to a change of the Vmax of Ca2+ transport (C: 9.7 +/- 1.6 nmol/mg/min; H: 5.4 +/- 1.4 nmol/mg/min). This is then followed by the decreased calcium channel receptor binding. However, the rate and the extent of depression in Ca2+-ATPase activity are much greater than that of Ca2+ channel receptor binding. Since alteration of Ca2+-ATPase is accompanied by an increase in intracellular Ca2+ concentration and there is a temporal association with the onset of myocardial lesions in the hypertensive rats, it is suggested that elevated intracellular calcium concentration as a result of altered Ca2+-ATPase activity may play a significant role in the development of hypertensive cardiomyopathy.

Rat heart-derived endothelial and smooth muscle cell cultures: isolation, cloning and characterization.

This report describes the initiation, cloning and establishment of long-term serial cultures of rat heart-derived vascular endothelial (EC) and smooth muscle cells (SMC). Populations of these cells derived from both the macro-and microcirculation were obtained utilizing isolated heart perfusion technique. Elimination of potential mesothelial cell contamination was achieved by ethanol fixation of the pericardial surface prior to perfusion. Initial outgrowths from perfusate yielded both endothelial (rapid adhering) and smooth muscle (slow adhering) appearing cell populations. Subsequent pooling of individual EC colonies resulted in maintaining, with gradual subcultivation, a stable homogeneous population which was designated RHE-parent. Upon continual subculture late passage (greater than P10) RHE-parent cell cultures expressed a marked heterogeneity in endothelial phenotypes. Cloning experiments resulted in establishing two distinct EC populations designated RHE-clone 1A and RHE-clone 2A. All RHE cell cultures exhibited the typical cobblestone growth pattern and positive immunofluorescent staining for factor VIII related antigen. In contrast, rat heart-derived smooth muscle cell (RH-SMC) cultures displayed the typical multilayered 'hill and valley' pattern and positive fluorescence for SMC-specific actin and myosin antibodies. Additional EC preparations, obtained without prior fixation of the pericardial surface, revealed cell clusters which stained positive for cytokeratin. On the other hand, RHE parent and cloned populations stained exclusively for vimentin, further confirming the absence of mesothelial cell contamination in these cultures. Cell growth studies on early (less than P10) and late (greater than P10) passage RHE-parent population revealed markedly different cell growth responses and cell morphology. Both EC cloned populations and more notably RHE-parent (less than P10) cultures were capable of significant growth when maintained in limiting serum concentration. Growth studies using serum-free RHE-parent conditioned medium demonstrated mitogenic activity when tested on RHE-parent cultures indicating the presence of an endothelial cell-derived growth factor. These studies indicate that long-term RHE and RH-SMC derived cell cultures can serve as a useful model to study the biology of vascular cells derived from different sites. In addition the demonstration of mitogenic activity in these cultures will enable us to explore further the nature of this response and compare this phenomenon with growth factors identified in large vessel cell systems.

Rat cerebral microvascular smooth muscle cells in culture.

This report describes the development and establishment of long-term serial cultures of adult rat vascular smooth muscle cells (SMC) derived from cerebrocortical resistance vessels (small arteries and arterioles). Electron microscopic examination of microvessels isolated off a 150 microns nylon mesh sieve clearly demonstrated the predominance of these vessel types. Initial outgrowth from collagenase-elastase-treated microvessel fragments yielded both endothelium and smooth muscle cells. However, at confluency (2-3 weeks) these cultures consisted of a homogeneous population of broad, polygonal cells that grew in a multilayered "hill and valley" pattern typical of SMC in vitro. For comparative morphological and functional studies, SMC cultures were also initiated from rat thoracic aortas utilizing ring segments as explants. The smooth muscle origin of cultures derived from both resistance vessel (RV) and aorta (RA) was further demonstrated by positive immunofluorescent staining by the specific smooth muscle alpha-actin and myosin antibodies. Ultrastructural examination of these SMC cultures revealed similar morphologic features consisting of typical cytoplasmic myofilament bundles with associated dense bodies and numerous pinocytotic vesicles. Cell growth studies on early (less than P 15)- and late (greater than P 15)-passage RV- and RA-SMC populations revealed markedly different cell growth responses. Representative growth curves of early- and late-passage RA-SMC showed a significantly higher growth rate (two- to fourfold) than RV-SMC cultures. Both cultures, however, exhibited a marked increase in growth potential at higher passage levels. Heparin, at a concentration of 100 micrograms/ml inhibited the growth of RV-SMC during the first 3 days after addition in both exponential and growth-arrested culture states, whereas RA-SMC cultures showed no inhibitory response. These studies indicate that long-term RV-SMC cultures can serve as a useful model system to study functional and metabolic properties of this cell type and provide the means to explore further the heterogeneity of SMC derived from different vasculatures in normal as well as various disease states.