Adipose tissue is the main energy reserve of the body. When energy is required, adipocyte triglycerides stored in lipid droplets (LDs) are broken down by lipase, and free fatty acids are released to supply the physiological need. Intracellular LDs are active metabolic organelles in mammalian cells, particularly in adipocytes. The present study was aimed to investigate the morphological changes of LDs and the alternation of LD-associated perilipin family proteins during long-term lipolysis stimulated by forskolin. Primary differentiated adipocytes derived from epididymal fat pads of Sprague-Dawley (SD) rats were incubated in the presence or absence of 1 μmol/L forskolin for 24 h. Content of glycerol released to the culture medium was determined by a colorimetric assay and served as an index of lipolysis. Morphological changes of LDs were observed by Nile red staining. The mRNA level of perilipin family genes was detected by quantitative real-time PCR. The protein level and subcellular localization were examined by immunoblotting and immunofluorescence staining, respectively. The results showed that forskolin induced sustained lipolysis in differentiated adipocytes. The morphology of LDs changed in a time-dependent manner. Large clustered LDs became gradually smaller in size and eventually disappeared; in contrast, peripheral micro-LDs increased gradually in number until the cytoplasm was filled with numerous micro-LDs. The protein level of the perilipin family proteins showed obvious alternation. Mature adipocytes physiologically expressed a very low level of Plin2 protein, whereas in adipocytes stimulated with lipolytic forskolin, the protein and mRNA levels of Plin2 were significantly increased, and the increased Plin2 was specifically bound to the surface of LDs. During chronic stimulation of forskolin, the mRNA level of Plin3 was unchanged, but the mRNA levels of Plin1, Plin4 and Plin5 were significantly decreased. These results suggest that the morphology of LDs and perilipin family proteins on the surface of LDs are significantly altered during long-term lipolysis stimulated by forskolin, representing a dynamic process of the remodeling of LDs.
Adipocytes ; drug effects ; Animals ; Cells, Cultured ; Colforsin ; pharmacology ; Lipid Droplets ; Lipolysis ; Perilipin-2 ; metabolism ; Perilipins ; metabolism ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo observe the effect of Kanli Granule (KG) on myocardial mechanics in pressure overload induced diastolic heart failure (DHF) rats.

METHODSTotally 60 male Wistar rats were divided into the sham-operation group, the model group, the KG group, and the Valsartan group according to random digit table, 15 in each group. The pressure overload induced DHF model was established in all groups except the sham-operation group using abdominal aortic constriction surgery. Totally 7 rats died after modeling (with the mortality of 10. 67%) , and the rest 53 finished the following test. Rats in the KG group were administered with KG extract (calculated as 6. 75 g crude drug/kg) by gastrogavage. Rats in the Valsartan group were administered with Valsartan (7.2 µg/g) by gastrogavage. Equal volume of double distilled water was administered to rats in the model group and the sham-operation group by gastrogavage. All rats were intervened for 32 weeks. The response of isolated heart papillary muscle tonus to isoprenaline (ISO) and adenylate cyclase (Forskolin) was respectively observed. The enhancement phenomenon after resting development force (DF) of isolated heart papillary muscle tonus, and changes of DF in different Ca²⁺ concentrations were observed.

RESULTS(1) In the ISO response test: Compared with the sham-operation group, the amplifications of DF, ±df/dt, -df/dt were obviously elevated in the model group (P < 0.05). Compared with the model group, the amplifications of DF and ±df/dt were obviously lowered in the KG group (P < 0.01), and the amplification of ±df/dt was also reduced in the Valsartan group (P < 0.01). (2) In the Forskolin response test: Compared with the sham-operation group, the amplifications of DF and ±df/dt obviously increased in the model group (P < 0.05). Compared with the model group, the amplifications of DF and ±df/dt were obviously reduced in the KG group (P < 0.01), and the amplification of DF was also reduced in the Valsartan group (P < 0.05). (3) In post-resting DF enhancement test: Compared with the sham-operation group, the amplification of DF showed gradually decreasing tendency along with prolonged resting time in the model group, and they were obviously lowered at all time points (P < 0.05). Compared with the model group, the amplification of DF was gradually increasing along with prolonged resting time in the KG group. The amplification of DF at post-resting 240 s was obviously larger in the KG group than in the model group (P < 0.05). The amplification of post-resting DF still showed gradually decreasing tendency along with prolonged resting time in the Valsartan group, with increased amplifications of DF at post-resting 60 s and 120 s (P < 0. 05) (4) The amplifications of DF in different Ca²⁺ concentrations: Compared with the sham-operation group, the amplifications of DF were significantly elevated in different Ca²⁺ concentrations (1.75, 3.5, 7.0 mmol/L ) (P < 0.05, P < 0.01). Compared with the model group, there was no statistical difference in amplification of DF in different Ca²⁺ concentrations in the KG group (P > 0.05). The amplifications of DF in different Ca²⁺ concentrations were significantly reduced in the Valsartan group (P < 0.05).

CONCLUSIONSThe ISO response and the Forskolin response were enhanced in isolated heart papillary muscle tonus of pressure overload induced DHF rats; enhanced post-resting DF was reduced; DF in different supra-physiologic levels of Ca²⁺ was still enhanced. KG could significantly improve excessive enhancement of pressure overload induced DHF rats in ISO response and Forskolin response, and improve enhancement of post-resting myocardium.

Animals ; Colforsin ; pharmacology ; Drugs, Chinese Herbal ; pharmacology ; Heart ; drug effects ; physiopathology ; Heart Failure, Diastolic ; drug therapy ; Isoproterenol ; pharmacology ; Male ; Random Allocation ; Rats ; Rats, Wistar

AIMTo study the influences of dibutyryl cyclic adenosine monophosphate (dbcAMP) and forskolin on human sperm motility in vitro.

METHODSSemen samples, aseptically obtained by masturbation and prepared by swim-up technique from 20 fertile men, were incubated with different concentrations of dbcAMP and forskolin at 37 deg. Measurements were carried out after 10 min, 20 min, 30 min and 60 min incubation. Motility parameters were estimated by using an automatic analyzing system.

RESULTSTreatment with dbcAMP or forskolin resulted in a significant increase in sperm motility and progressive motility. The larger the concentrations of dbcAMP or forskolin, the greater the effect appeared. The straight linear velocity and curvilinear velocity were not affected by both agents.

CONCLUSIONdbcAMP and forskolin increase the motility and progressive motility of human sperm in vitro.

Adult ; Bucladesine ; administration & dosage ; pharmacology ; Colforsin ; administration & dosage ; pharmacology ; Dose-Response Relationship, Drug ; Humans ; In Vitro Techniques ; Male ; Osmolar Concentration ; Sperm Motility ; drug effects

OBJECTIVETo investigate the role of cysteinyl leukotriene (CysLT) receptors in the differentiation of rat glioma C6 cells.

METHODSRat glioma C6 cells were treated with the agonist LTD(4), the CysLT(1) receptor antagonist montelukast and the differentiation inducer forskolin. Cell morphology and GFAP protein expression were determined after treatments.

RESULTForskolin (10 μmol/L) induced morphological changes and GFAP protein expression (cell differentiation) in C6 cells, but LTD(4) (0.1-100 nmol/L) did not induce these changes. Montelukast (1 μmol/L) alone did not affect C6 cell differentiation, while it induced the differentiation when combined with the LTD(4) (100 nmol/L).

CONCLUSIONThe CysLT(2) receptor may modulate the differentiation of rat glioma C6 cells.

Acetates ; pharmacology ; Animals ; Cell Differentiation ; drug effects ; Cell Line, Tumor ; Colforsin ; pharmacology ; Cysteine ; Glioma ; metabolism ; pathology ; Leukotriene Antagonists ; pharmacology ; Leukotriene D4 ; pharmacology ; Leukotrienes ; Quinolines ; pharmacology ; Rats ; Receptors, Leukotriene ; agonists

Cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-dependent chloride channel, plays key roles in fluid secretion in serous epithelial cells. Previously, we identified two polymethoxylated flavonoids, 3',4',5,5',6,7-hexamethoxyflavone (HMF) and 5-hydroxy-6,7,3',4'-tetramethoxyflavone (HTF) which could potentiate CFTR chloride channel activities. The present study was aimed to investigate the potentiation effects of HMF and HTF on CFTR Cl(-) channel activities by using a cell-based fluorescence assay and the short circuit Ussing chamber assay. The results of cell-based fluorescence assay showed that both HMF and HTF could dose-dependently potentiate CFTR Cl(-) channel activities in rapid and reversible ways, and the activations could be reversed by the CFTR blocker CFTRinh-172. Notably, HMF showed the highest affinity (EC50 = 2 μmol/L) to CFTR protein among the flavonoid CFTR activators identified so far. The activation of CFTR by HMF or HTF was forskolin (FSK) dependent. Both compounds showed additive effect with FSK and 3-Isobutyl-1-methylx (IBMX) in the activation of CFTR, while had no additive effect with genistein (GEN). In ex vivo studies, HMF and HTF could stimulate transepithelial Cl(-) secretion in rat colonic mucosa and enhance fluid secretion in mouse trachea submucosal glands. These results suggest that HMF and HTF may potentiate CFTR Cl(-) channel activities through both elevation of cAMP level and binding to CFTR protein pathways. The results provide new clues in elucidating structure and activity relationship of flavonoid CFTR activators. HMF might be developed as a new drug in the therapy of CFTR-related diseases such as bronchiectasis and habitual constipation.
Animals ; Colforsin ; Colon ; metabolism ; Cystic Fibrosis Transmembrane Conductance Regulator ; drug effects ; Flavones ; physiology ; Flavonoids ; pharmacology ; Genistein ; Intestinal Mucosa ; metabolism ; Mice ; Rats

OBJECTIVETo investigate the duration of tau hyperphosphorylation and spatial memory retentive deficit induced by single injecting with Forskolin, a protein kinase A activator, into lateral ventricle of rats, and the correlation between the two pathological alterations.

METHODSForskolin (80 micromol/L, 40 microl) was injected into the lateral ventricle by stereotaxic injection. Tau phosphorylation and spatial memory retention were measured by Western blot/immunocytochemistry and Morris-Water-Maze test, respectively.

RESULTSThe phosphorylation levels of tau at Tau-1, PHF-1, and pS214 epitopes were significantly elevated at 24, 48 and 72 h after single administration of Forskolin (P < 0.05). The most significant elevation was seen at 48 h (P < 0.01) and it tended to recover at 72 h (P < 0.05) after injection. The correlation between the two pathological alterations was positive at PHF-1 site (r = 0.97, P < 0.05), negative at Tau-1 site (r = -0.963, P < 0.05), and not significant at pS214 site (r = 0.705, P > 0.05).

CONCLUSIONSForskolin can induce tau hyperphosphorylation and spatial memory retentive deficit within a certain period of time. The level of tau phosphorylation in hippocampus is somehow correlated with the spatial memory deficit in rats.

Animals ; Colforsin ; pharmacology ; Injections, Intraventricular ; Lateral Ventricles ; Male ; Memory Disorders ; chemically induced ; Phosphorylation ; Random Allocation ; Rats ; Rats, Wistar ; Time Factors ; tau Proteins ; metabolism

The aim of the present study was to investigate the effects of cyclic adenosine monophosphate (cAMP) on rat gastric antral circular smooth muscle function. Forskolin, a direct activator of adenylyl cyclase (AC), was used to observe the influences of cAMP. Multi-channel physiological recorder was used to record spontaneous contraction activity of gastric antral circular muscle from Wistar rats. And ELISA method was used to detect the change of cAMP production in perfusate. The results showed that forskolin concentration-dependently suppressed the amplitude and frequency of the spontaneous contraction of the gastric antral muscle, and lowered the baseline of contraction movement significantly. Forskolin concentration-dependently increased the production of cAMP in the perfusate, which showed a significant negative correlation with the contraction amplitude of gastric antral ring muscle. The inhibitory effect of forskolin on spontaneous contraction activity of rat gastric antral circular muscle could be blocked by cAMP-dependent protein kinase (PKA) inhibitor H-89. These results suggest forskolin increases cAMP production and then activates PKA pathway, resulting in the inhibition of the spontaneous contraction activity of rat gastric antral circular smooth muscle.
Adenylyl Cyclases ; metabolism ; Animals ; Colforsin ; pharmacology ; Cyclic AMP ; pharmacology ; Cyclic AMP-Dependent Protein Kinases ; metabolism ; Isoquinolines ; pharmacology ; Muscle, Smooth ; drug effects ; Pyloric Antrum ; drug effects ; Rats ; Rats, Wistar ; Sulfonamides ; pharmacology

BACKGROUNDCyclic adenosine monophosphate (cAMP) could activate chloride channels in bovine ciliary body and trigger an increase in the ionic current (short-circuit current, Isc) across the ciliary processes in pigs. The purpose of this study was to investigate how cAMP modulates Isc in isolated human ciliary processes and the possible involvement of chloride transport across the tissue in cAMP-induced Isc change.

METHODSIn an Ussing-type chamber system, the Isc changes induced by the cAMP analogue 8-bromo-cAMP and an adenylyl cyclase activator forskolin in isolated human ciliary processes were assessed. The involvement of Cl(-) component in the bath solution was investigated. The effect of Cl(-) channel (10 µmol/L niflumic acid and 1 mmol/L 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS)), K(+) channel (10 mmol/L tetraethylammonium chloride (TEA)), or Na(+) channel blockers (1 mmol/L amiloride) on 8-bromo-cAMP-induced Isc change was also studied.

RESULTSDose-dependently, 8-bromo-cAMP (10 nmol/L-30 µmol/L) or forskolin (10 nmol/L-3 µmol/L) increased Isc across the ciliary processes with an increase in negative potential difference on the non-pigmented epithelium (NPE) side of the tissue. Isc increase induced by 8-bromo-cAMP was more pronounced when the drug was applied on the NPE side than on the pigmented epithelium side. When the tissue was bathed in low Cl(-) solutions, the Isc increase was significantly inhibited. Finally, niflumic acid and DIDS, but not TEA or amiloride, significantly prevented the Isc increase induced by 8-bromo-cAMP.

CONCLUSIONScAMP stimulates stroma-to-aqueous anionic transport in isolated human ciliary processes. Chloride is likely to be among the ions, the transportation of which across the tissue is triggered by cAMP, suggesting the potential role of cAMP in the process of aqueous humor formation in human eyes.

8-Bromo Cyclic Adenosine Monophosphate ; pharmacology ; Chloride Channels ; antagonists & inhibitors ; Ciliary Body ; drug effects ; physiology ; Colforsin ; pharmacology ; Cyclic AMP ; physiology ; Humans ; In Vitro Techniques ; Potassium Channel Blockers ; pharmacology ; Sodium Channel Blockers ; pharmacology

We isolated mouse embryonic cardiomyocytes derived from timed-pregnant females at different periods and used patch-clamp technique to investigate the muscarinic cholinergic modulation of pacemaker current I(f) in different developmental stages. In early development stage (EDS), muscarinic agonist carbachol (CCh) significantly decreased the magnitude of the pacemaker current I(f) but had no effect in late development stage (LDS). Forskolin (a direct adenylate cyclase activator) and IBMX (a non-selective phosphodiesterase inhibitor) increased I(f) in both EDS and LDS cells. Interestingly, although both forskolin and IBMX increased basal I(f), their effects on CCh-inhibited I(f) were different. Forskolin did not reverse the inhibitory action of CCh until intermediate development stage (IDS). In contrast, IBMX reversed the inhibitory action of CCh on I(f) in EDS but not in IDS. It is suggested that a decrease in intracellular cAMP is a possible mechanism for CCh to modulate I(f). During the EDS and IDS CCh controls the cytoplasmic cAMP level by different pathways: In EDS, CCh modulates I(f) possibly by activating PDE which accelerates the breakdown of cAMP, but in IDS possibly by inhibiting adenylate cyclase (AC) which then reduces the synthesis of cAMP.
Animals ; Carbachol ; pharmacology ; Colforsin ; metabolism ; pharmacology ; Female ; Heart ; embryology ; physiology ; Mice ; Muscarinic Agonists ; pharmacology ; Myocytes, Cardiac ; drug effects ; physiology ; Pacemaker, Artificial ; Phosphodiesterase Inhibitors ; metabolism ; pharmacology ; Pregnancy ; Receptors, Muscarinic ; metabolism

The short-circuit current (I(SC)) technique was used to examine the effects of cAMP-evoking agents, forskolin/IBMX, and a Chinese medicinal formula, Huoxiang-zhengqi liquid (HZL) on HCO(3)(-) secretion by intact porcine distal airway epithelium. The freshly isolated airway epithelial tissue displayed a transepithelial basal current of (94.9±8.2) μA/cm(2), 16.6% and 62.7% of which was inhibited by amiloride (epithelial Na(+) channel blocker, 100 μmol/L) and NPPB (cystic fibrosis transmembrane conductance regulator Cl(-) channel blocker, 100 μmol/L). Substitution of Cl(-) with impermeable gluconate(-) in the K-H bath solution resulted in a basal current of (54.0±6.7) μA/cm(2), which could be abolished by further removal of HCO(3)(-) in the solution, indicating HCO(3)(-) secretion under unstimulated conditions. Application of forskolin/IBMX (10 μmol/L/100 μmol/L) stimulated an increase of (13.8±1.9) μA/cm(2) in I(SC) which could be blocked by Cl(-) channel inhibitor DPC. With Cl(-) and Cl(-)/HCO(3)(-) substitution, forskolin/IBMX evoked an increase of (7.3±0.5) μA/cm(2) in HCO(3)(-)-dependent, DPC-inhibitable I(SC) (I(HCO(3))). Noticeably, basolateral application of HZL (10 μL/mL) in normal K-H solution evoked an I(SC) of (15.9±2.4) μA/cm(2). The EC(50) of this I(SC) was (6.1±1.4) μL/mL. When substituting Cl(-), HZL stimulated an increase of (7.4±1.9) μA/cm(2) in I(HCO(3)), suggesting HZL-induced HCO(3)(-) secretion. After pretreating the epithelial tissues with forskolin/IBMX in Cl(-)-free K-H solution, HZL induced a further increase of (8.4±0.9) μA/cm(2) in I(HCO(3)), and pretreating tissues with HZL did not significantly affect the subsequent forskolin/IBMX-induced I(HCO(3)) response, indicating that HZL- and forskolin/IBMX-induced I(HCO(3)) responses appeared to be independent and be most likely mediated via different cellular mechanisms. Our results suggest that HCO(3)(-) can be secreted by porcine distal airway epithelium under unstimulated and stimulated conditions, and the stimulatory effect of HZL on HCO(3)(-) secretion in the distal airway epithelium shows HZL to be a hopeful new agonist for distal airway HCO(3)(-) secretion that could be of therapeutic significance.
Amiloride ; pharmacology ; Animals ; Bicarbonates ; metabolism ; Biological Transport ; Colforsin ; pharmacology ; Cystic Fibrosis Transmembrane Conductance Regulator ; antagonists & inhibitors ; Drugs, Chinese Herbal ; pharmacology ; Epithelium ; drug effects ; metabolism ; Respiratory System ; drug effects ; metabolism ; Swine