Adhesion-related kinase induction of migration requires phosphatidylinositol-3-kinase and ras stimulation of rac activity in immortalized gonadotropin-releasing hormone neuronal cells.

GnRH neurons migrate into the hypothalamus during development. Although migratory defects may result in disordered activation of the reproductive axis and lead to delayed or absent sexual maturation, specific factors regulating GnRH neuronal migration remain largely unknown. The receptor tyrosine kinase, adhesion-related kinase (Ark) (also known as Axl, UFO, and Tyro7), has been implicated in the migration of GnRH neuronal cells. Binding of its ligand, growth arrest-specific gene 6 (Gas6), promotes cytoskeletal remodeling and migration of NLT GnRH neuronal cells via Rac and p38 MAPK. Here, we examined the Axl effectors proximal to Rac in the signaling pathway. Gas6/Axl-induced lamellipodia formation and migration were blocked after phosphatidylinositol-3-kinase (PI3K) inhibition in GnRH neuronal cells. The p85 subunit of PI3K coimmunoprecipitated with Axl and was phosphorylated in a Gas6-sensitive manner. In addition, PI3K inhibition in GnRH neuronal cells diminished Gas6-induced Rac activation. Exogenous expression of a dominant-negative form of Ras also decreased GnRH neuronal lamellipodia formation, migration, and Rac activation. PI3K inhibition blocked Ras in addition to Rac activation and migration. In contrast, pharmacological blockade of the phospholipase C gamma effectors, protein kinase C or calcium/calmodulin protein kinase II, had no effect on Gas6/Axl signaling to promote Rac activation or stimulate cytoskeletal reorganization and migration. Together, these data show that the PI3K-Ras pathway is a major mediator of Axl actions upstream of Rac to induce GnRH neuronal cell migration.

The permeability transition pore triggers Bax translocation to mitochondria during neuronal apoptosis.

Cerebellar granule neurons (CGNs) require depolarization for their survival in culture. When deprived of this stimulus, CGNs die via an intrinsic apoptotic cascade involving Bim induction, Bax translocation, cytochrome c release, and caspase-9 and -3 activation. Opening of the mitochondrial permeability transition pore (mPTP) is an early event during intrinsic apoptosis; however, the precise role of mPTP opening in neuronal apoptosis is presently unclear. Here, we show that mPTP opening acts as an initiating event to stimulate Bax translocation to mitochondria. A C-terminal (alpha9 helix) GFP-Bax point mutant (T182A) that constitutively localizes to mitochondria circumvents the requirement for mPTP opening and is entirely sufficient to induce CGN apoptosis. Collectively, these data indicate that the major role of mPTP opening in CGN apoptosis is to trigger Bax translocation to mitochondria, ultimately leading to cytochrome c release and caspase activation.

Radiation-related pericarditis.

To determine the incidence of pericardial effusion in patients undergoing upper mantle radiation therapy, 81 patients with Hodgkin's disease, stages I to IIIB, were selected from a protocol series of 98 patients. Twenty-four patients (29.6 percent) met X-ray criteria for the presence of pericardial effusion. Eleven of the 24 also underwent right heart catheterization to confirm the presence of pericardial effusion and to define any hemodynamic abnormality. Fourteen patients were found to have transient effusion. Five of the 11 patients have had partial pericardiectomy for symptoms and signs of cardiac tamponade. There has been no evidence of recurrent Hodgkin's disease in these surgically treated patients. Ninety-two percent of the pericardial effusions occurred in the first 12 months after the end of radiation therapy. Therapeutic implications depend on elucidation of the natural history of this process. At present close follow-up is necessary with surgical intervention for signs or symptoms of cardiac tamponade.

Systolic time intervals before and after maximal exercise treadmill testing for evaluation of chest pain.

The change in systolic time intervals from before exercise to three to four minutes following a maximal-exercise treadmill test was measured to eveluate chest pain in 110 fasting supine subjects. Forty-six (85 percent) of 54 patients with chest pain and with abnormal findings on coronary arteriograms were found to have at least a 10-msec prolongation in the left ventricular ejection time index (LVETI), whereas only two (8 percent) of 25 subjects without heart disease and 5 (16 percent) of 31 subjects with chest pain but with normal findings on coronary arteriograms had 10 msec or more of prolongation of the LVETI after exercise. The change in the other systolic time intervals (total electromechanical systole, preejection phase [PEP], and PEP/LVET) were less reliable in detecting the presence or absence of coronary disease. We conclude that determination of LVETI before and after maximal-exercise treadmill testing is a clinically useful noninvasive disgnostic test for obstructive coronary disease in patients with chest pain.

Endoplasmic reticulum stress and trophic factor withdrawal activate distinct signaling cascades that induce glycogen synthase kinase-3 beta and a caspase-9-dependent apoptosis in cerebellar granule neurons.

Loss of trophic or activity-dependent survival signals is commonly recognized as a stimulus for neuronal apoptosis and may play a significant role in neurodegeneration. Recent data have also implicated endoplasmic reticulum (ER) stress as an important factor in some neurodegenerative conditions. However, whether shared or unique apoptotic cascades are activated by trophic factor withdrawal (TFW) versus ER stress in primary neurons has not previously been investigated. In primary cultures of rat cerebellar granule neurons (CGNs), the ER stressor brefeldin A activated a discrete pathway involving the following: (1) stimulation of the ER resident kinase PERK, (2) enhanced phosphorylation of the translation initiation factor eIF2alpha, and (3) increased expression and nuclear localization of the transcription factor Gadd153/CHOP. ER stress-induced CGN apoptosis was blocked by an antagonist of IP3 receptor-mediated Ca2+ release, 2-aminoethoxydiphenyl borate (2-APB), and by expression of ER-targeted Bcl-2. In contrast, CGN apoptosis elicited by TFW (i.e., removal of serum and depolarizing extracellular potassium) did not display any ER stress component nor was it blocked by either 2-APB or ER-Bcl-2. Despite these apparent differences, both brefeldin A and TFW induced dephosphorylation (activation) of glycogen synthase kinase-3beta (GSK-3beta). Moreover, inhibitors of GSK-3beta (IGF-I, lithium) and caspase-9 (LEHD-fmk) significantly protected CGNs from apoptosis induced by either ER stress or TFW. These data indicate that ER stress and TFW elicit distinct signals that activate GSK-3beta and intrinsic apoptosis in neurons.

Proteasome inhibition elicits a biphasic effect on neuronal apoptosis via differential regulation of pro-survival and pro-apoptotic transcription factors.

The role of the proteasome in neuronal apoptosis is poorly understood since both anti- and pro-apoptotic effects result from proteasome inhibition. We studied the effects of proteasome inhibition in cultured rat cerebellar granule neurons. Acute exposure to proteasome inhibitors MG-132 and lactacystin blocked caspase activation induced by removal of depolarizing medium. However, chronic treatment with MG-132 activated caspases in neurons maintained in depolarizing potassium. The biphasic effect of MG-132 was hypothesized to be due to differential degradation of anti- and pro-apoptotic proteins. Accordingly, acute exposure to MG-132 inhibited the hyperphosphorylation, loss of DNA binding, ubiquitination, and degradation of the pro-survival transcription factor MEF2D induced by removal of depolarizing medium. In contrast, chronic exposure to MG-132 increased the expression and phosphorylation of c-Jun, elevated levels of the pro-apoptotic protein Bim, and triggered neuronal apoptosis, even in the presence of depolarizing medium. Thus, proteasome inhibition exerts an acute pro-survival action by stabilizing MEF2 transcription factors. However, chronic proteasome inhibition causes a build-up of phosphorylated c-Jun and Bim, which eventually overwhelms the effects of MEF2 and triggers apoptosis.

Coronary artery status of apparently healthy subjects with frequent and complex ventricular ectopy.

Twenty-five subjects from a cohort of 62 asymptomatic, apparently healthy subjects incidentally discovered to have frequent and complex ventricular ectopy were studied with cardiac catheterization and coronary angiography. Fourteen had normal coronary arteries, five noncritical coronary artery disease (less than 50% luminal narrowing), and six significant coronary artery disease (greater than or equal to 50% luminal narrowing). Slightly elevated left ventricular end diastolic pressures were found in all subject subgroups. Characteristics of the ventricular ectopy detected by maximal exercise testing or 24-hour Holter ambulatory electrocardiography did not differentiate those subjects with coronary artery disease from those with normal coronary arteries. This study documents that a minority of apparently healthy subjects with frequent and complex ventricular arrhythmia have significant coronary artery disease and supports a conservative approach to the management of such patients.

Systemic heparinization during percutaneous coronary angiography: evaluation of effectiveness in decreasing thrombotic and embolic catheter complications.

Systemic heparinization has been advocated as preventive for thrombotic and embolic complications of arterial catheterization. To test this hypothesis, 95 patients undergoing coronary angiography via the percutaneous femoral arterial approach were randomized into heparinized and nonheparinized groups. Evaluation for thrombotic and embolic complications by clinical means and non-invasive electrical impedance flow measurements in the lower limbs was performed precatheterization, postcatherization, and at 4 and 24 hr. Clinical data reveal loss of distal leg pulses in 11% (5/74) of the nonheparinized group, with two of these individuals developing signs of claudication and requiring embolectomy. No individuals (0/48) in the heparinized group lost distal leg pulses. Immediate, 4-hr, and 24-hr post-catheterization bloodflow was 12%, 10%, and 12% lower, respectively, in the catheterized limb of those in the nonheparinized group. At 24 hr 52% of the nonheparinized group had bloodflow levels lower than the precatheterization levels in the right (catheterized) extremity, while 2% (2/48) of the heparinized group had a similar reduction. One possible complication of excess bleeding was noted with heparin. It is concluded that systemic heparinization is safe and can be an important adjunct in the reduction of thromboembolic complications of percutaneous coronary angiography.

Objective evidence of occult myocardial dysfunction in patients with frequent ventricular ectopy without clinically apparent heart disease.

Eighteen asymptomatic persons without apparent cardiac disease were incidentally discovered to have frequent ventricular ectopic activity (VEA) (more than a mean of 100 b/hr during 24-hour ambulatory ECG examination) and were found by cardiac catheterization to have normal coronary arteriograms. Thirteen persons (72%) also demonstrated complex (multiform or repetitive patterns) VEA and eight persons were found to have undiagnosed hypertension. Examination of left ventricular (LV) angiographic and hemodynamic data of these persons showed elevated LV end-systolic volume index in 10 persons (56%), elevated LV and end-diastolic volume index in 12 persons (67%) and elevated LV end-diastolic pressure in 11 persons (61%). Although ejection fractions of all but three persons were normal, impaired myocardial contractility, as measured by decreased mean velocity of circumferential fiber shortening (less than 1.0 circ/sec), was found in 10 persons (56%). Abnormalities of LV function were more prevalent in persons with higher mean frequencies of VEA (more than 300 b/hr), but did not seem related to the presence of complex VEA. Etiologic mechanisms of the frequent and complex VEA could not be defined. We conclude that subclinical evidence of myocardial dysfunction is present in some persons without apparent cardiac disease who have frequent VEA as evidence by subtle abnormalities of increased LV volumes and end-diastolic pressure and decreased mean velocity of myocardial circumferential fiber shortening.