WYKŁAD

NO- sytazy

nNOS(1), eNOS(2),

iNOS(3)



receptor

GTP ATP

CG – cGMP CA - cAMP

PKG PKA

VASP

PDE 1 - 8

Serce, mózg

mięśniówka

gładka

ANP BNP NO

GTP

pCG sCG

cGMP cGMP

Hamowanie Rozkrcz m.

gładkiej

czynności

m. sercowego

i m. szkieletowych

Serce, mózg

mięśniówka

gładka

Receptory



adrenergiczne



ATP ATP

pCA sCA

cAMP cAMP

Pobudzenie Ca Rozkurcz m. gładkiej

czynności

m. sercowego

i m. szkieletowych

NO – syntaza śródbłonka

eNOS

Czynniki rozprzęgające dipol

(eNOS – eNOS)

Cukrzyca

Nadciśnienie

Miażdżyca

Chroniczne palenie

Tolerancja na nitraty

NO/cGMP kaskada sygnałów.

NO endogenny syntetyzowany przez NO- syntazy: eNOS i
nNOS

Nitraty - uwalniane z egzogennie stosowanych donatorów

NO - aktywuje CG i prowadzi do wzrostu poziomu cGMP.



cGMP jako II-przekaźnik moduluje

aktywność

 cGMP -zależnych kinaz,

 kanałów jonowych bramkowanych przez cGMP,

 cGMP -regulowanych fosfodwuesteraz.

Wymienione efektory są ściśle związane z licznymi
funkcjami fizjologicznymi

 układu sercowo – naczyniowego

 układu nerwowego.

 YC-1 and BAY 58-2667 reprezentują nową klasę aktywatorów
CG
- aktywowanej przez NO.

Nowy mechanizm regulacji naczyń związany z

hamowaniem cGMP- zależnej kinazy proteinowej (PKG).

Peptyd DT-2 penetrujący błonę komórkową – inhibitor

PKG.



Konstytutywna aktywność PKG wywiera ciągłe modulujące działanie

na
toniczne napięcie naczyń wyzwalane nawet przy minimalnym poziomie

cGMP.



Wzrost poziomu cGMP powoduje dalszy wzrost aktywności kinazy



(High PKG Activity), nasilając wpływ modulujący tego enzymu.



Dostępne inhibitory, takie jak :



KT-5823 (strzałka niebieska)



pochodne Rp-cGMPS (strzałka zielona),



oraz Rp-8-pCPT-cGMPS i Rp-8-Br-PET-cGMPS,

odwracają aktywność kinazy PKG tylko do podstawowego

poziomu.



Pochodne Rp-cGMPS wywierają także częściowo stymulujące działanie na

kinazy
co przedstawia podwójna strzałka.



DT-2 (czerwona strzałka), hamuje nie tylko aktywność PKG stymulowanej

przez
cGMP ale także podstawową aktywność PKG, znosząc w ten sposób

regulację
naczyniową związaną z kinazą.

Identyfikacja VASP jako fosfoproteiny stymulującej
wazodylatację

(Vasodilator-stimulated phosphoprotein) oraz ustalenie,
że jest ona

substratem



dla cGMP- zależnej kinazy proteinowej (PKG)

 Seryna 239 jest miejscem preferencyjnym dla
fosforylacji
przez PKG

 dla cAMP- zależnej kinazy proteinowej (PKA)

 Seryna 157 jest miejscem preferencyjnym dla
fosforylacji
przez PKA .

Funkcja VASP jako czynnika regulującego

proliferację komórek mięśniówki gładkiej SMC.

VASP spełnia funkcję modulatora wzrostu komórek
mięśniówki gładkiej poprzez indukowanie sygnałów
pozytywnych i negatywnych związanych z różnymi
miejscami fosforylacji VASP.

 Fosforylacja VASP seryny 157 przy współudziale
PKA wyzwala
proliferację,

 Fosforylacja VASP indukowana przez NO przy
współudziale
PKG w miejscu seryny 239 hamuje proliferację.

Molekularne mechanizmy kreują

pogląd, że inhibitory PDE5 mają

działanie:

kardioprotekcyjne,

- antyhypertroficzne

- hamują remodeling
komórek mięśnia sercowego i
naczyń.

 obniżają wewnątrzkomórkowy poziom wapnia i redukują
skurcz
naczyń i mięśnia sercowego

 wykazują działanie antyhyperteroficzne związane z
obciążeniem
następczym antihypertrophic response to pressure
overload

 mito = mitochondrion;

ch = channel;

NPs = natriuretic peptides;

SR = sarcoplasmic reticulum;

PKC = protein kinase C.

Scheme of nitric oxide (NO) metabolism pathway. In this
diagram the nitric oxide (NO) pathway is depicted.

In presence of oxygen (O2) and/or alveolar ventilation, NO
synthases (NOS) are activated and produce NO from L-
arginine via L-citrulline.

The NO activates soluble- and membrane-bound guanylate
cyclases, which synthesize cyclic guanylate monophosphate
(cGMP), which subsequently activates cGMP-kinase.

This enzyme—by activation of K+-channels and
subsequent Ca++-channel inhibition—evokes a reduction of
intracellular Ca++ concentration, finally resulting in
vasodilation.

The downstream effects of NO are limited by
phosphodiesterase (PDE)-induced degradation of cGMP.

Recently, the results of a double-blind, placebo-controlled
multicenter study demonstrated that daily inhaled iloprost
significantly improved exercise capacity, New York Heart
Association (NYHA) functional classification, dyspnea
scoring, and event-free survival over a three-month
observation period in patients with selected forms of PAH
and chronic thromboembolic PH.

Olschewski H, Simonneau G, Galie N, et al. Inhaled iloprost
for severe pulmonary hypertension. N Engl J Med.
2002;347:322–329.

cAMP= cyclic adenylate monophosphate
cGMP= cyclic guanylate monophosphate
GC= guanylate cyclase
HIV= human immunodeficiency virus
NO= nitric oxide  NYHA= New York Heart Association
PAH= pulmonary arterial hypertension
PDE= phosphodiesterase
PH= pulmonary hypertension
PPH= primary pulmonary hypertension

Hemodynamic and gas-exchange response to inhaled
nitric oxide (NO), infused PGI2, and oral sildenafil in
patients with lung fibrosis and pulmonary hypertension.

Deviations from preintervention baseline are displayed
for inhaled NO, infused prostacyclin (PGI iv), and oral
sildenafil (Sil oral).

CO = cardiac output;
mPAP = mean pulmonary arterial pressure;
mSAP = mean systemic arterial pressure;
PaO 2 = partial pressure of arterial oxygen (changes in
mm Hg);
PVRI = pulmonary vascular resistance index;
PVR/SVR ratio = ratio of pulmonary to systemic vascular
resistance

(Ghofrani et al., Lancet 2002;360:895–900).

The NO/cGMP axis represents a pivotal signaling pathway for
the lung circulation.

Phosphodiesterases, as regulators of the second messenger
response to endogenous NO, are thus of great therapeutic
potential for the treatment of lung circulatory disorders.

Among the clinically available PDE inhibitors, sildenafil is a
most promising agent for pulmonary vasodilation and long-
term antiremodeling in the lung vasculature of PAH patients.

Although orally administered, sildenafil does possess features
of pulmonary selectivity.

It may be favorably combined with other vasodilative and
antiproliferative agents.

Large trials, including a placebo-controlled phase III trial with
sildenafil in patients with PAH, are currently underway.

Intracellular signaling pathway of nitric oxide (NO),
prostanoids, and natriuretic peptides: role of
phosphodiesterases (PDEs).

Ligands (i.e., NO, atrial natriuretic peptide [ANP], brain
natriuretic peptide [BNP], and prostanoids) activate
membrane-bound or soluble cyclases.

Guanylate and adenylate cyclases generate cyclic
guanosine monophosphate (cGMP) and adenosine
monophosphate (cAMP) from GTP and ATP.

These intracellular second messengers, via activation of
protein kinases, induce cellular responses (i.e.,
vasodilation and anti-proliferation).

Phosphodiesterases limit the effects of the ligands by
degradation of second messengers cGMP and cAMP into
inactive GMP and AMP.

Thus, by inhibition of the PDEs, the PDE inhibitors augment
and prolong the cellular responses to NO, prostanoids, and
natriuretic peptides.

The present study is the first to compare the short-
termhemodynamic profiles of three different PDE5 inhibitors—
sildenafil, vardenafil and tadalafil—in a well-defined patient
collective suffering from chronic PAH.

Although vardenafil showed the most rapid onset of effects, this
substance lacked selectivity for the pulmonary circulation, which
was demonstrated for sildenafil and tadalafil, even at high doses
of the latter agent.

The pulmonary vasodilatory response to tadalafil appeared to be
the most long-lasting, as anticipated from previous studies in the
field of erectile dysfunction.

By contrast with sildenafil, vardenafil and tadalafil did not
improve arterial oxygenation.
Obviously, we cannot translate short-term effects into long-term
effects, because we know that long-term effects may be
significantly more efficacious than short-term effects, such as has
been observed with epoprostenol; however, the converse may also
occur.

These findings thus strongly support the notion that careful
evaluation of the pulmonary hemodynamic and gas exchange
effects of each new PDE inhibitor focusing on cGMP decay is to be
undertaken, despite common classification as PDE5 inhibitors.

The pharmacologic mechanisms involving sildenafil and cGMP

at the vascular tissue/platelet interface.

EC = endothelial cells;

GMP = guanosine monophosphate, a catabolized product of

cGMP;

sGC = soluble guanylate cyclase;

VSMC = vascular smooth-muscle cells.

Sildenafil,

vardenafil, and tadalafil inhibitory PDE 5

 W sposób zależny od stężenia powoduje rozkurcz mięśniówki
gładkiej tętnic i powoduje wzrost stężenia cGMP i
pozostaje bez wpływu na stężenie cAMP.

Usunięcie śródbłonka powoduje aż 45 krotne przesunięcie krzywej
dla sildenafilu i 21 krotne dla tadalafilu oraz aż 251 krotne dla
vardenafilu.

Maksymalna reakcja na sildenafil i tadalafil ulega odpowiednio
redukcji do 38 % i 53%. Maksymalna reakcja na vardenafil nie ulega
zmianie

Inhbitory NO – syntazy i inhibitory cyklazy guanylanowej i w
zmiatacze NO powoduje także obniżenie rozkurczowego działania
inhibitorów FDE 5.

Sildenafil, tadalafil i vardenafil potęgują znacznie rozkurczowe
działanie nitratów naturetycznego peptydu.

Sildenafil, tadalafil i vardenafil hamują skurcze naczyń wyzwalane
przez
CaCl

(0.01-5

mM) i fenylefrynę.

Ouabain, kw. cyclopiazonowy i Kalykulin nie wpływają na
wazodylatacyjne dzialanie inhibitorów FDE 5.

Wyniki te wskazują że tylko vardenafil, zmienia

handling in the

rat aorta in addition to increasing cGMP

levels through inhibition of

PDE5 to cause relaxation.

Model of NOS3-PDE5A coregulation of ß-adrenergic stimulated

contractility in the heart. In a wild-type (WT) normal myocyte

(left),

ß-adrenergic stimulation triggers adenylate-cyclase (AC)-

coupled activation of cAMP and NOS3/sGC generation of

cGMP.

cAMP enhances intracellular calcium and activates PKA to

enhance contractility.

cGMP signaling is compartmentalized by regional placement of

PDE5A, which targets the cGMP-PKG activity to a region

strategically linked to adrenergic regulation.

Acute PDE5A inhibition enhances localized cGMP and PKG

activity to blunt adrenergic stimulation.

Acute NOS3 blockade by L-NAME removes the required cGMP

substrate and thus efficacy of PDE5A inhibition, but exogenous NO

can still restore efficacy.

With genetic lack of NOS3 or with chronic NOS inhibition (right),

PDE5A moves away from z-bands (left).

This removes its physiologic regulation of acute

adrenergic signaling, and exogenous NO can no longer restore

the efficacy of PDE5A inhibition.

Diagram przedstawia komórkowe mechanizmy
pośredniczące w zależnym od czasu ekspozycji inotropowo -
ujemnym działaniu IL- 6 ARVM (in a time-dependent
manner)

Ekspozycja na 2 – 24 godzinne dzialanie IL-6 indukuje
zależne od iNOS/ NO - inotropowo ujemne działanie w którym
pośredniczy
JAK2/STAT3 (Yu et al. 2003, 2005).

Podczas wczesnej fazy działania (2 godzinnej ekspozycji na IL-6
dochodzi
do stymulacji sCG/ cGMP/PKG, która ma być odpowiedzialna za
działanie
inotropowo – ujemne.

Wydłużeniu ekspozycji do 24 godzin IL-6 wyzwala dwa mechanizmy

powoduje obniżenie kurczliwości

 oraz wyzwala hamownie funkcji SR

Schematyczny model potencjalnych mechanizmów

oddziaływania NO i prostacyclin (PGI2) na

działanie CO, wyzwalające proces hyperpolaryzacji

rozkurcz

mięśniówki gładkiej naczyń

AC - cyklaza adenylanowa; GC - cyklaza

guanylanowa; IP- prostacyclin receptor;

KCa, aktywowane Ca2+- kanały K+ .

Model of vascular smooth muscle PKG activity and its impact on
vascular tone. We propose that constitutive activity of PKG (Basal
PKG Activity) exerts continuous modulation of vascular tone even at
minimal cGMP levels.
Increased cGMP further stimulates the kinase (High PKG Activity),
enhancing its modulatory influence.

Available inhibitors, including KT-5823 (blue arrow) and the Rp-cGMPS
derivatives (green arrow), such as Rp-8-pCPT-cGMPS and Rp-8-Br-PET-
cGMPS, reverse cGMP-stimulated PKG activity to varying degrees, but
only toward basal levels.

Rp-cGMPS derivatives may also yield partial kinase stimulation as
indicated by the double-headed arrow. DT-2 (red arrow), on the other
hand, inhibits not only the cGMP-stimulated PKG activity but also a
substantial portion of the basal PKG activity, essentially abolishing
vasoregulation by the kinase.

Scheme showing possible cross talk points of interaction between cyclic
nucleotides (cNTs (cAMP and cGMP)) and the IP

-Ca

-, Ca

-CaM-MLCK-,

MAP kinase-, Rho-kinase-, PKC-, and myosin phosphatase pathways in
smooth muscle.

Upon activation of smooth muscle by activation of the GPCRs through
Ca

-mobilizing agonists the [Ca

]

increases due to an influx of Ca

through plasmalemmal Ca

channels or IP

-mediated release from the

SR.
Ca

binds to CaM and activates MLCK to phosphorylate MLC

resulting in contraction. Myosin is dephosphorylated by myosin
phosphatase.
Activation of G

-coupled receptors inhibits adenylate cyclases,

decreasing cAMP and inactivating PKA (not shown in the scheme).
Activation of GPCRs also leads to stimulation of cPLA

and the release

of AA for prostaglandin (PG) biosynthesis.

Activation of receptor tyrosine kinases leads to the stimulation of PLC
and Ras and subsequently to proliferative response. In addition to the
MLCK pathway, which comprises the major pathway in muscle
contraction, MLC

can also be directly phosphorylated by Rho-kinase,

PKC, and p42/p44 MAPK.

Inhibition of myosin phosphatase by AA, PKC, or Rho-kinase increases
MLC

phosphorylation and contraction. Several targets for cNTs

regulation have been reported, including the receptor, G proteins,
PLCß, IP

receptor, the Ca

-pump in the SR , MLCK, Rho-kinase, MAP

kinase, myosin phosphatase, and the plasmalemmal Ca

channel.

Cross talk between the cNTs and these targets could constitute the
biochemical mechanisms underlying relaxation in smooth muscle.
Abbreviations are the same as given in the text; (+) and (-), stimulation
and inhibition, respectively.

Model of vascular smooth muscle PKG activity and its impact on
vascular tone. We propose that constitutive activity of PKG
(Basal PKG Activity) exerts continuous modulation of vascular
tone even at minimal cGMP levels.

Increased cGMP further stimulates the kinase (High PKG
Activity), enhancing its modulatory influence.

Available inhibitors, including KT-5823 (blue arrow) and the Rp-
cGMPS derivatives (green arrow), such as Rp-8-pCPT-cGMPS and
Rp-8-Br-PET-cGMPS, reverse cGMP-stimulated PKG activity to
varying degrees, but only toward basal levels.

Rp-cGMPS derivatives may also yield partial kinase stimulation
as indicated by the double-headed arrow. DT-2 (red arrow), on
the other hand, inhibits not only the cGMP-stimulated PKG
activity but also a substantial portion of the basal PKG activity,
essentially abolishing vasoregulation by the kinase.

Schematic representation of the NO/cGMP/cGKI pathway of
vascular smooth muscle relaxation and mechanisms of its
inactivation by endothelial dysfunction. Also illustrated is the
analysis of NO/cGMP/cGKI action and endothelial function by
monitoring cGKI phosphorylation of Ser239 of the substrate
protein VASP. Under normal conditions, nitric oxide (NO),
synthesized by nitric oxide synthase (NOS) III, stimulates soluble
guanylate cyclase (sGC), increasing cGMP and thus stimulation of
cGMP-dependent protein kinase I (cGKI) and vasorelaxation.
This pathway can, however, be inhibited at several sites.
Interventions that reduce vascular oxidative stress such as
therapy with statins, Ang II receptor (AT1 subtype) blockers, ACE
inhibitors, or vitamins improves NO bioavailability by reducing
vascular superoxide production. In all cases, P-VASP served as a
reliable monitor of the NO/cGMP/cGKI pathway and endothelial
function.

Angiotensin II, hypertension, hypercholesterolemia, and
nitrate tolerance enhance vascular superoxide (O2·-)
production, which inactivates NO, diminishing cGKI action.

Both O2·- and the NO/O2·- reaction product peroxynitrite
(ONOO-) potently inhibit sGC.

Peroxynitrite can also uncouple NOSIII by oxidizing the
NOSIII cofactor tetrahydrobiopterin (BH4) to
dihydrobiopterin (BH2) and/or by oxidizing Zn thiolate
complexes within NOSIII.

Angiotensin II and nitroglycerin (NTG) therapy stimulate
the activity and expression of phosphodiesterase PDE1A1,

thus decreasing cGKI action and endothelium-dependent

and -independent vasodilation

while inducing supersensitivity to vasoconstrictors.

Mechanisms of cGKI inhibition of Ca2+ release (left) and Ca2+
sensitization (right) to inhibit myosin light chain (MLC)
phosphorylation and SMC contraction.

cGKI phosphorylation (P) of IRAG inhibits IP3/IRAG-mediated Ca2+
release from the endoplasmic reticulum (ER), thus inhibiting MLC
kinase (MLCK) and vasoconstriction.

cGKI phosphorylates Rho and interferes with Rho kinase inhibition of
MLCP, thus MLCP dephosphorylates MLC and enhances SMC
relaxation.

Binding of cGKI to substrates (eg, IRAG and the myosin-binding
subunit [MBS] of MLCP), which are also members of signaling
complexes or scaffolds, could be an important aspect of achieving
specificity of cGKI action.

G indicates G-protein; IP3R, IP3 receptor; NO, nitric oxide; PLC,
phospholipase C; and sGC, soluble guanylate cyclase.

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