[Ca2+]i transients by reverse mode of cardiac Na+/Ca2+ exchanger (NCX1) were recorded in fura-2 loaded BHK cells with stable expression of NCX1. Repeated stimulation of reverse NCX1 produced a long-lasting decrease of Ca2+ transients ('rundown'). Rundown of NCX1 was independent of membrane PIP2 depletion. Although the activation of protein kinase C (PKC) was observed during the Ca2+ transients, neither a selective PKC inhibitor (calphostin C) nor a PKC activator (PMA) changed the degrees of rundown. By comparison, a non-specific PKC inhibitor, staurosporine (STS), reversed rundown in a dose-dependent and reversible manner. The action of STS was unaffected by pretreatment of the cells with calphostin C, PMA, or forskolin. Taken together, the results suggest that the stimulation of reverse NCX1 by STS is independent of PKC and/or PKA inhibition.
Forskolin ; Fura-2 ; Membranes ; Naphthalenes ; Protein Kinase C ; Staurosporine

To elucidate the mechanism of cyclic nucleotides, such as adenosine 3',5'-cyclic monophosphate (cAMP) and guanosine 3',5' -cyclic monophosphate (cGMP), in the regulation of human gastric motility, we examined the effects of forskolin (FSK), isoproterenol (ISO) and sodium nitroprusside (SNP) on the spontaneous, high K+ and acetylcholine (ACh)-induced contractions of corporal circular smooth muscle in human stomach. Gastric circular smooth muscle showed regular spontaneous contraction, and FSK, ISO and SNP inhibited its phasic contraction and basal tone in a concentration-dependent manner. High K+ (50 mM) produced sustained tonic contraction, and ACh (10 micrometer) produced initial transient contraction followed by later sustained tonic contraction with superimposed phasic contractions. FSK, ISO and SNP inhibited high K+-induced tonic contraction and also ACh-induced phasic and tonic contraction in a reversible manner. Nifedipine (1 micrometer), inhibitor of voltage-dependent L-type calcium current (VDCC(L)), almost abolished ACh-induced phasic contractions. These findings suggest that FSK, ISO and SNP, which are known cyclic nucleotide stimulators, inhibit smooth muscle contraction in human stomach partly via inhibition of VDCCL.
Acetylcholine ; Adenosine ; Calcium ; Contracts ; Forskolin ; Guanosine ; Humans ; Isoproterenol ; Muscle, Smooth ; Nifedipine ; Nitroprusside ; Nucleotides, Cyclic ; Relaxation ; Stomach

This study was designed to clarify the mechanism of the inhibitory effect of forskolin on contraction, cytosolic Ca2+ level ([Ca2+]i), and Ca2+ sensitivity in guinea pig ileum. Forskolin (0.1 nM~10 micrometer) inhibited high K+ (25 mM and 40 mM)- or histamine (3 micrometer)-evoked contractions in a concentration-dependent manner. Histamine-evoked contractions were more sensitive to forskolin than high K+-evoked contractions. Spontaneous changes in [Ca2+]i and contractions were inhibited by forskolin (1 micrometer) without changing the resting [Ca2+]i. Forskoln (10 micrometer) inhibited muscle tension more strongly than [Ca2+]i stimulated by high K+, and thus shifted the [Ca2+]i-tension relationship to the lower-right. In histamine-stimulated contractions, forskolin (1 micrometer) inhibited both [Ca2+]i and muscle tension without changing the [Ca2+]i-tension relationship. In alpha-toxin-permeabilized tissues, forskolin (10 micrometer) inhibited the 0.3 micrometer Ca2+-evoked contractions in the presence of 0.1 mM GTP, but showed no effect on the Ca2+-tension relationship. We conclude that forskolin inhibits smooth muscle contractions by the following two mechanisms: a decrease in Ca2+ sensitivity of contractile elements in high K+-stimulated muscle and a decrease in [Ca2+]i in histamine-stimulated muscle.
Animals ; Contracts ; Cytosol ; Forskolin ; Guanosine Triphosphate ; Guinea ; Guinea Pigs ; Histamine ; Ileum ; Muscle Tonus ; Muscle, Smooth ; Muscles

BACKGROUND: Propofol may decrease myocardial contractility via actions on the beta-adrenoceptor-mediated signal transduction. The aim of this study was to evaluate the effect of propofol via beta-adrenoceptor-mediated signal transduction by measuring the tissue levels of cAMP (cyclic adenosine monophosphate). METHODS: The effects of propofol on beta-adrenoceptor mediated cascades were measured with cAMP concentrations, which were stimulated by agonists (l-isoproterenol, GTPgammaS, and forskolin) of each step of beta-adrenoceptor-mediated cascades. RESULTS: While the production of cAMP stimulated by isoproterenol, GTPgammaS, or forskolin are increased (P < 0.05), application of each concentration of propofol (0.1, 1, 10, 100 micrometer) did not alter the levels of cAMP. CONCLUSIONS: Considering that propofol did not alter the tissue cAMP levels when stimulated by isoproterenol, GTPgammaS, and forskolin, propofol appears to have no effect on the beta-adrenoceptor signaling pathway in guinea pig ventricular myocardium.
Adenosine ; Animals ; Forskolin ; GTP-Binding Proteins ; Guanosine 5'-O-(3-Thiotriphosphate) ; Guinea Pigs ; Isoproterenol ; Myocardium ; Propofol ; Signal Transduction

The terminal differentiation of malignant melanoma cells is known to be induced by activating cAMP signaling pathway with alpha-MSH or cAMP analogues. However, sustained activation of cAMP signaling system that induces the differentiation of melanoma cells, also induces the desensitization of the pathway at the receptor level. Nevertheless, the adaptation of adenylate cyclase (AC) expression by sustained activation of cAMP signaling system has not been clearly understood. This study was performed to examine whether the sustained activation of cAMP system induce changes in the expression AC isoforms as an adaptation mechanism. Treatment of B16/F10 murine melanoma cells with 100 mM forskolin for 6 days resulted in differentiation, melanin accumulation and increased expression of tyrosine hydroxylase mRNA. In the forskolin-treated melanoma cells, change in expression of various AC isoform at the transcription level was detected by reverse-transcription polymerase chain reaction (RT-PCR). Expression of AC isoform mRNA: ACI, III, VI, VII, and IX increased to the level of 196-392% of the control whereas the level of ACII was decreased by 30%. The cAMP concentration was increased both in basal and alpha-MSH stimulated cells, but the AC activity was decreased in the forskolin treated cells. Thus, these results suggest that sustained activation of cAMP system induces differential expression of AC isoforms, which results in increase of cAMP accumulation.
Adenylate Cyclase/*genetics ; Animal ; Cell Differentiation ; Cyclic AMP/*metabolism ; Forskolin/*pharmacology ; Isoenzymes/genetics ; Melanoma, Experimental/*enzymology/*pathology ; Mice ; Signal Transduction

Forskolin is a complex labdane plant diterpenoid, which has been used in the treatment of a variety of diseases based on its activity as an activator of adenosine monophosphate(cAMP) cyclase. Natural forskolin exists only in the cork layer of the root of Coleus forskohlii. Due to the complexity of the extraction and chemical synthesis processes, the yield and purity of forskolin cannot meet commercial requirements. In recent years, with the rapid development of synthetic biology and the analysis and interpretation of many diterpene biosynthetic pathways, a new approach has been provided for the green production of forskolin. In this paper, the structure, activity, biosynthetic pathway and the heterologous biosynthesis of forskolin were reviewed. The problems and solutions in the heterologous biosynthesis of forskolin were also discussed and summarized, which will provide references for the construction of high-yielding forskolin engineering strains.
Biosynthetic Pathways ; Colforsin

The effect of forskolin on corticostriatal synaptic transmission was examined by recording excitatory postsynaptic currents (EPSCs) in rat brain slices using the whole-cell voltage-clamp technique. Forskolin produced a dose-dependent increase of corticostriatal EPSCs (1, 3, 10, and 30micrometer) immediately after its treatment, and the increase at 10 and 30micrometer was maintained even after its washout. When the brain slices were pre-treated with (DL)-2-amino-5-phosphonovaleric acid (AP-V, 100micrometer), an NMDA receptor antagonist, the acute effect of forskolin (10micrometer) was blocked. However, after washout of forskolin, an increase of corticostriatal EPSCs was still observed even in the presence of AP-V. When KT 5720 (5micrometer), a protein kinase A (PKA) inhibitor, was applied through the patch pipette, forskolin (10micrometer) increased corticostriatal EPSCs, but this increase was not maintained. When forskolin was applied together with AP-V and KT 5720, both the increase and maintenance of the corticostriatal EPSCs were blocked. These results suggest that forskolin activates both NMDA receptors and PKA, however, in a different manner.
Animals ; Brain ; Carbazoles ; Cyclic AMP-Dependent Protein Kinases ; Excitatory Postsynaptic Potentials ; Forskolin ; N-Methylaspartate ; Patch-Clamp Techniques ; Pyrroles ; Rats ; Receptors, N-Methyl-D-Aspartate ; Synaptic Transmission

Skeletal muscle contains several precursor cells that generate muscle, bone, cartilage and blood cells. Although there are reports that skeletal muscle-derived cells can trans-differentiate into neural-lineage cells, methods for isolating precursor cells, and procedures for successful neural induction have not been fully established. Here, we show that the preplate cell isolation method, which separates cells based on their adhesion characteristics, permits separation of cells possessing neural precursor characteristics from other cells of skeletal muscle tissues. We term these isolated cells skeletal muscle-derived neural precursor cells (SMNPs). The isolated SMNPs constitutively expressed neural stem cell markers. In addition, we describe effective neural induction materials permitting the neuron-like cell differentiation of SMNPs. Treatment with retinoic acid or forskolin facilitated morphological changes in SMNPs; they differentiated into neuron-like cells that possessed specific neuronal markers. These results suggest that the preplate isolation method, and treatment with retinoic acid or forskolin, may provide vital assistance in the use of SMNPs in cell-based therapy of neuronal disease.
Animals ; Antigens, Differentiation/metabolism ; Cell Adhesion ; *Cell Differentiation ; Cell Lineage ; Cell Separation ; Cells, Cultured ; Forskolin/pharmacology ; Mice ; Mice, Inbred ICR ; Muscle, Skeletal/*cytology/metabolism ; Neurons/*cytology/metabolism ; Stem Cells/*cytology/metabolism ; Tretinoin/pharmacology

PURPOSE: Erectile dysfunction (ED) remains a major complication from cavernous nerve injury during radical prostatectomy. Recently, stem cell treatment for ED has been widely reported. This study was conducted to investigate the availability, differentiation into functional cells, and potential of human muscle-derived stem cells (hMDSCs) and human adipose-derived stem cells (hADSCs) for ED treatment. MATERIALS AND METHODS: We compared the neural differentiation of hMDSCs and hADSCs. Human muscle and adipose tissues were digested with collagenase, followed by filtering and centrifugation. For neural induction, isolated hMDSCs and hADSCs were incubated in neurobasal media containing forskolin, laminin, basic-fibroblast growth factor, and epidermal growth factor for 5 days. Following neural induction, hMDSCs and hADSCs were differentiated into neural cells, including neurons and glia, in vitro. RESULTS: In neural differentiated hMDSCs (d-hMDSCs) and differentiated hADSCs (d-hADSCs), neural stem cell marker (nestin) showed a significant decrease by immunocytochemistry, and neuronal marker (beta-tubulin III) and glial marker (GFAP) showed a significant increase, compared with primary hMDSCs and hADSCs. Real-time chain reaction analysis and Western blotting demonstrated significantly elevated levels of mRNA and protein of beta-tubulin III and GFAP in d-hADSCs compared with d-hMDSCs. CONCLUSIONS: We demonstrated that hMDSCs and hADSCs can be induced to undergo phenotypic and molecular changes consistent with neurons. The neural differentiation capacity of hADSCs was better than that of hMDSCs.
Adipose Tissue ; Blotting, Western ; Caves ; Cell Differentiation ; Centrifugation ; Collagenases ; Epidermal Growth Factor ; Erectile Dysfunction ; Forskolin ; Humans ; Immunohistochemistry ; Laminin ; Male ; Muscles ; Neural Stem Cells ; Neuroglia ; Neurons ; Prostatectomy ; RNA, Messenger ; Stem Cells ; Tubulin

BACKGROUND AND OBJECTIVES: Reports of neural differentiation of mesenchymal stem cells suggest the possibility that these cells may serve as a source for stem cell-based regenerative medicine to treat neurological disorders. The purpose of this study was to generate neural cells by differentiation of bone marrow-derived mesenchymal stem cells that isolated from human mastoid process. MATERIALS AND METHOD: Human mesenchymal stem cells (hMSCs) isolated from human mastoid process bone marrow during mastoidectomy for chronic otitis media surgery were characterized using fluorescence-activated cell sorter. Induction of neural differentiation from hMSCs was performed using mitogenic factors (basic fibroblast growth factor, epidermal growth factor, forskolin, isobutylmethylxanthine), and the characterization of differentiated hMSCs was performed using immunohistochemistry, RT-PCR and whole cell patch clamp technique. RESULTS: hMSCs from bone marrow of mastoid process were isolated and cultured. Differentiated cells from hMSCs expressed mRNA transcripts for neuron specific markers, TUJ1 and neurofilament proteins (NF-L, NF-M) as determined by RT-PCR, and neuron specific markers, suhc as NeuN, TUJ1, microtubule-associated protein-2 (MAP2) and glial fibrillary acidic protein by immunohistochemistry. These cells showed voltagedependent sodium currents that was blocked by tetrodotoxin. CONCLUSION: hMSCs, which were isolated from human mastoid process bone marrow, were one of the good sources for stem cell-based regenerative medicine to treat neurological disorders.
Bone Marrow ; Cell Differentiation ; Epidermal Growth Factor ; Fibroblast Growth Factors ; Forskolin ; Glial Fibrillary Acidic Protein ; Humans ; Immunohistochemistry ; Mastoid ; Mesenchymal Stromal Cells ; Nervous System Diseases ; Neurofilament Proteins ; Neurons ; Otitis Media ; Regenerative Medicine ; RNA, Messenger ; Sodium ; Temporal Bone